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Applied and Environmental Microbiology, August 2001, p. 3702-3706, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3702-3706.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of a Granulovirus Isolated from Epinotia
aporema Wals. (Lepidoptera: Tortricidae) Larvae
Alicia
Sciocco-Cap,1
Alejandro D.
Parola,2,3
Alina V.
Goldberg,1
Pablo D.
Ghiringhelli,2 and
Víctor
Romanowski2,3,*
Instituto de Microbiología y
Zoología Agrícola, CNIA-INTA, CC 25, 1712 Castelar1; Departamento de Ciencia y
Tecnología, Universidad Nacional de Quilmes, 1976 Bernal2; and Instituto de
Bioquímica y Biología Molecular, Facultad de Ciencias
Exactas, Universidad Nacional de La Plata, 1900 La
Plata3, Argentina
Received 8 January 2001/Accepted 11 May 2001
 |
ABSTRACT |
A granulovirus (GV) isolated from Epinotia aporema
(Lepidoptera: Tortricidae)
a major soybean pestwas studied in terms
of its main morphological, biochemical, and biological properties. The
ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the
granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly
described granulovirus (EpapGV) indicate that it is
indeed a good candidate for the biological control of this pest.
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TEXT |
The bean shoot borer,
Epinotia aporema (Wals.), is a serious pest of
soybean and other legume crops in South America. In Argentina, damages
caused by this tortricid decrease soybean yields up to 40%, depending
on population level and environmental conditions (18).
Currently, broad-spectrum chemical insecticides are used to control
this caterpillar, delaying or suppressing field colonization by
natural enemies. This scenario compromises the implementation of a
proper integrated pest management scheme for soybean crops, in areas
where the incidence of E. aporema reaches high population levels (25).
Among pathogens, a granulovirus (GV) was detected as the main mortality
factor (6, 26). In general, the potential of baculoviruses
for pest control has been well documented and they have proven to be
effective against many pests (9, 15, 24). In this regard,
preliminary experiments showed that E. aporema GV
(EpapGV) could be a safe alternative for inclusion in soybean pest management (25). However, former studies were
directed towards evaluating its virulence, and none of the previously
cited reports addressed the characterization of the virus. As an
initial point in our research, we identified and characterized a GV
isolate from E. aporema, indigenous to the main soybean area
of Argentina.
Virus isolation and multiplication.
A colony of E. aporema caterpillars reared on artificial medium (12)
was established in our laboratory. EpapGV was isolated from a
single E. aporema larva collected in Oliveros (Santa Fe, Argentina). Viral amplification was carried out, allowing fourth-instar larvae to feed on formalin-free artificial diet, superficially contaminated with 4,000 granules per mm2.
Granules were purified from homogenized larvae by two cycles of
centrifugation on continuous 40 to 65% (wt/wt) sucrose gradients at
100,000 × g for 1 h. Virions were released by
hydrolyzing granules in 0.1 M
Na2CO3-0.17 M NaCl-0.01 M
EDTA (pH 10.5) at 37°C for 30 min. Following the dissolution of the
granules, the suspension was neutralized by addition of 1 M HCl, and
undissolved material was removed by low-speed centrifugation.
Electron microscopy.
Tissues were dissected from infected and
control larvae, fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.3)-0.25 M sucrose for 3 h, postfixed in 1% osmium
tetroxide for 1 h, dehydrated through an ethanol-propylene oxide
series, and embedded in Epon-Araldite resin. Ultrathin sections,
stained in 2% uranyl acetate and lead citrate, were examined with a
JEM-1200 EX II electron microscope.
The occlusion bodies were ovoidal in shape, with a mean size of 466 (±8.9) by 296 (±6.0) nm. In general, each granule contained a single
rod-shaped virion (290 [±10.9] × 61.2 [±1.8] nm) with one
nucleocapsid (226 [±8.9] × 41.7 [±4.1] nm) (Fig.
1A and B). In rare cases, granules
containing more than one virion were observed (Fig. 1C).
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FIG. 1.
Electron micrographs illustrating the major
morphological properties of EpapGV. (A) Section through an
infected epidermal cell showing virions and granules (bar, 1 µm). (B)
Longitudinal section through an occlusion body with a single embedded
virion. Note the "nipple" and the "claw" ends of the
nucleocapsid (bar, 100 nm). (C) Granule with multiple embedded virions
(bar, 100 nm).
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Major occlusion body protein.
The major protein components of
the occlusion bodies were analyzed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (21).
Proteins were visualized after staining with Coomassie brilliant blue,
and sizes were estimated using molecular weight markers (Sigma, St.
Louis, Mo.). The estimated molecular mass of the EpapGV major
protein band (28.5 ± 0.5 kDa) (Fig.
2A) falls within the 27- to 31-kDa range,
characteristic for granulins (27, 36).
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FIG. 2.
(A) SDS-polyacrylamide gel electrophoresis of
EpapGV proteins. Granules were purified from infected larvae
as described in the text and run on a 12% polyacrylamide gel. Lane 1, molecular mass markers; lane 2, granules from EpapGV; lane 3, granules from CpGV. (B) Autoradiography of EpapGV DNA
restriction fragments. Viral DNA was digested with EcoRI
(E), BamHI (B), HindIII (H), and
BglII (Bg); labeled with [ -32P]dATP;
and resolved by electrophoresis on an 0.4% agarose gel. Lambda DNA was
digested with HindIII (M1). (C) The same samples as in panel
B separated on a 1.5% agarose gel. Lambda DNA digested with
BamHI, HindIII, and BglII
(M2) and pcDNA II DNA (Invitrogen) digested with
HindIII and HinfI (M3) were used as
molecular size markers (indicated in base pairs).
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For amino acid sequencing, the proteins were resolved in a
Tricine-polyacrylamide gel system (
30) and transferred to
a polyvinylidene
difluoride membrane for 2 h at 0.25 A, and the
granulin was visualized
by Ponceau red staining (1% in water for 15 min and destaining
with water). The section of membrane containing
granulin (approximately
20 µg) was cut out and processed for protein
sequencing by a modified
Edman degradation method (
23) at
the LANAIS-PRO laboratory (Buenos
Aires,
Argentina).
The N-terminal amino acid sequence of the protein
(GYNKSLRYSRHEGTT) was compared with that of the homologous
proteins from
other GVs and in most cases showed no more difference
than 1 or
2 out of 15 residues. The N-terminal sequences of polyhedrins
are less conserved and clearly distinct from the granulin consensus
sequence (Table
1). The lack of a
methionine residue at the amino
terminus probably reflects
posttranslational processing. This
is consistent with another granulin
sequence (
Agrotis segetum GV) that was also obtained by
protein sequencing instead of deduction
of the data from the DNA
sequence (
20).
Genome.
Virion suspensions were adjusted to a final
concentration of 0.5% SDS and incubated with proteinase K (0.25 mg/ml
at 37°C for 3 h), and the DNA was isolated by phenol
extraction and ethanol precipitation (29). The DNA
fragments obtained after digestion with BamHI,
BglII, EcoRI, and HindIII
endonucleases were separated by electrophoresis on 0.4 and 1.5%
(wt/vol) agarose gels, stained with ethidium bromide, and photographed
under UV light. Alternatively, DNA digests were labeled with
[-32P]dATP, separated by electrophoresis,
and visualized by autoradiography (29). The restriction
patterns of EpapGV DNA were distinct from those published for
other GVs (3, 4, 5, 7, 11, 14, 31, 33, 37, 39) (Fig. 2B
and C). The existence of multiple fragments of similar mobilities was
assessed by densitometry of the autoradiographs. To minimize the
inaccuracy associated with the determination of the sizes of very large
restriction fragments, double digests using combinations of restriction
endonucleases were also analyzed (data not shown). A size of 120 kbp
for the EpapGV genome was estimated from the sum of the
fragments (Fig. 2B and C). This result is consistent with the known
genomes of different GVs, which range from 89 kbp for Adoxophyes
orana GV (22) to 179 kbp for Xestia
c-nigrum GV (11). Detailed genome analysis is under
way to complete the physical restriction map and sequencing of
EpapGV, which would confirm the EpapGV species taxonomic status.
Since no submolar fragments were apparent in the restriction
endonuclease analyses, the EpapGV isolate studied in our
laboratory
seems to be homogeneous. However, further studies on the
viral
genome and the comparison of DNA digests from other geographical
and temporal isolates of EpapGV will allow us to determine if
there are variants of this virus, as previously described for
other GV
isolates (
3,
5,
32,
37).
Histopathology.
For light microscopy, larvae were fixed
in aqueous Bouin's solution for 48 h, dehydrated in a
tertiary-butyl alcohol series, and embedded in Paraplast, and
5-µm-thick sections were cut and stained using the modified Azan
technique (13). EpapGV infection was
polyorganotropic; the infected tissues included fatty tissue, tracheal matrix, and epidermis. Microscope observations of fatty tissue sections showed various stages of nuclear and cell
disintegration (Fig. 3A and B).
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FIG. 3.
Healthy (A) and infected (B) fatty tissue sections; the
infected tissue shows an intense red color throughout the cells due to
the presence of granules and a high degree of disintegration. By 3 days
postinfection, the symptoms of the disease are evident; the last-instar
control and infected larvae show differences in growth, development,
and color (C). The infected larvae turn yellowish and swollen, and
their development is interrupted, while the control larvae exhibit the
characteristic pink color of the prepupal stage. Shortly after death,
the larvae become brownish, and their tegument can be easily
ruptured (D).
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Biological activity.
The larvae infected by EpapGV
showed the typical symptoms of a GV infection at a late stage of the
disease, i.e., loss of appetite, decrease in mobility, and change of
color due to the accumulation of occlusion bodies in the infected
tissues (Fig. 3C). Shortly after death, the larvae became swollen and
turned brownish, and the tegument was easily disrupted (Fig. 3D).
In order to estimate the virulence of the isolate, bioassays were
conducted using the droplet feeding technique (
17). The
granules were suspended in a solution of 1% sucrose-0.1% Coomassie
brilliant blue (
1). The mean ingested volume per larva was
estimated for neonates (0.016 ± 0.001 µl) and fourth-instar
larvae
(2.4 ± 0.2 µl). On the basis of these measurements,
dilutions
were adjusted to obtain 5 doses of between 2 and 32 granules
per
larva for neonates and between 2 × 10
2
and 1.6 × 10
4 granules for fourth-instar
larvae in the 50% lethal dose (LD
50;
dose that
produces a lethal infection in 50% of the larvae in
7 days) assays.
Three replicate assays of 32 larvae each were
carried out for each
dose. An additional 32 larvae fed on the
colored sucrose solution were
used as
controls.
Those larvae which had ingested the virus suspension were transferred
individually to multiwell dishes containing formalin-free
diet and held
at 25 ± 1°C and 50% rH and in a 14-h-light/10-h-dark
cycle. Mortality was recorded every 6 h (neonates) or daily
(fourth-instar
larvae). The time-mortality response was determined
using the
data obtained with the lowest dose that yielded 100%
mortality.
Dose and time-mortality response data were analyzed using
ViStat
software (Cornell University, Ithaca, N.Y.).
The bioassays carried out with neonates and fourth-instar larvae
yielded LD
50 estimates of 5.6 (range, 3.64 to
7.34) and 4,000
(range, 3,880 to 5,200) granules per larva,
respectively. The
median survival time using the lowest dose that
yields 100% mortality
was 90 (±4.4) h for neonates and 105 (±16) h for fourth-instar
larvae. As expected, the time-mortality
response was dose dependent
(data not
shown).
Host specificity was studied using the percentage of mortality
and development of the disease as the criteria for
susceptibility.
The assays were performed on the major regional
soybean pests
in addition to
E. aporema, i.e.,
Anticarsia gemmatalis,
Rachiplusia nu, and
Spodoptera frugiperda (Lepidoptera: Noctuidae). Two
other
species, one distantly and one closely related,
Diatraea
saccharalis (Lepidoptera: Pyralidae) and
Cydia
pomonella (Lepidoptera: Tortricidae),
respectively, were also
included in the
assays.
Using the above-described method, doses from 4 to 256 granules per
larva were fed to neonates of each of these species. Doses
of up to 40 LD
50 for
E. aporema neonates did not
affect development
of the heterologous species. In all cases, with the
exception
of
C. pomonella, larvae fed on
EpapGV-contaminated diet pupated
simultaneously with the
controls. At the highest dose tested,
mortality produced by a GV was
observed only for
C. pomonella.
Subsequent restriction
enzyme analysis of the viral DNA, isolated
from the granules found in
dead larvae, indicated that the infection
was actually produced by a
C. pomonella GV (CpGV) (data not shown)
and was possibly due
to the activation of a latent virus present
in the
C. pomonella colony. This observation was previously reported
for
other baculoviral infections (
16).
EpapGV is a potential control agent for E.
aporema.
EpapGV has been considered as a
tentative species in the genus Granulovirus
(Baculoviridae) based on its pathology, although direct
morphological and biochemical characterization was lacking (38). Here, we present electron microscopy evidence and
biochemical data that confirm its generic identity. The biological
properties and DNA restriction patterns are consistent with a new GV
that seems to be different from others previously described in the literature.
According to the work of Federici (
10), three different
types of GVs can be distinguished by their unique pathobiological
pattern. In this regard, our results showed that EpapGV
exhibits
the typical broad tissue tropism of the type 2 GVs. This type
includes CpGV, which is the GV most successfully used for pest
control
(
9,
15). On the basis of the estimates for
LD
50 and
median survival time using the lowest
dose that yields 100% mortality
obtained for neonate larvae, it can be
concluded that EpapGV is
a highly virulent virus and, indeed,
a good candidate for the
microbial control of
E. aporema
larvae. Its potential is not limited
to soybean
a crop for which it
could be a complementary tool to
be used in combination with other
biotechnological or biological
strategies-but applies to the pest
management of other important
regional crops, such as beans, peanuts,
and alfalfa. However,
in order to develop a viral insecticide for
this pest, further
studies on its efficacy under field conditions are
necessary.
A significant decrease of the susceptibility with larval age was
apparent, a phenomenon that was observed elsewhere for several
other
baculoviruses (
2,
8,
19,
28,
34,
35). In this
context, it
is also important to establish complementary methods
to monitor the
population dynamics of
E. aporema, in order to
determine the
proper timing of application that would ensure control
during the early
instars of the larvae, when they are more exposed
and susceptible to
the
virus.
In summary, the results presented here provide the preliminary
information required both for further detailed characterization
of
EpapGV and for the development of a new alternative for
the
biocontrol of
E. aporema. In addition, it is interesting
that,
while environmentally sound pest management strategies are
available
for other insects that affect soybean,
E. aporema
is currently
controlled only with broad-spectrum chemical
insecticides.
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ACKNOWLEDGMENTS |
This research was supported by BID-SECyT 802 OC/AR PID 410 (V.R.
and A.S.-C.), UNLP X120 and X198 (V.R. and P.D.G.), and CIC BA (V.R.).
V.R. holds a research career award from CONICET (Consejo Nacional de
Investigaciones Científicas y Técnicas, Argentina).
We thank S. Lorenzatti de Diez and J. C. Gamundi (E.E.A. Oliveros
INTA, Argentina) for supplying the material from which the virus was
isolated and M. Rivas, D. Moreyra, and J. Cap for their collaboration
in insect rearing and bioassays.
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FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Bioquímica y Biología Molecular, Facultad de Ciencias
Exactas, Universidad Nacional de La Plata, calle 49 y 115, 1900 La
Plata, Argentina. Phone: 54-221-4250497, ext. 32. Fax: 54-221-4259223. E-mail: victor{at}biol.unlp.edu.ar.
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Applied and Environmental Microbiology, August 2001, p. 3702-3706, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3702-3706.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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