Biological Control 39 (2006) 96104

www.elsevier.com/locate/ybcon

Combined eects of azadirachtin and nucleopolyhedrovirus

(SpltNPV) on Spodoptera litura Fabricius
(Lepidoptera: Noctuidae) larvae
S. Senthil Nathan
a

a,b,*

, K. Kalaivani

Post Graduate and Research Department of Biotechnology, Vivekanandha College (W), Trichengode, Namakkal 637 205, Tamil Nadu, India
b
Plant Environment Division, Honam Agricultural Research Institute (HARI), National Institute of Crop Science (NICS),
Rural Development Administration (RDA), #381 Songhak-dong, Iksan, Chonbuk 570-080, Republic of Korea
c
Department of Zoology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India
Received 21 December 2005; accepted 22 June 2006
Available online 10 July 2006

Abstract
The eects of azadirachtin (AZA) and Spodoptera nucleopolyhedrovirus (SpltNPV) on development and mortality of Spodoptera
litura Fabricius (tobacco cutworm) were evaluated in the laboratory. The eective concentrations for AZA and SpltNPV were determined and tested as single and combination treatments. AZA and SpltNPV produced synergistic eects on tobacco cutworm mortality
in higher dose combination treatment. Combinations of AZA + SpltNPV at 0.25 ppm + 1 103 OB and 0.50 ppm + 1 106 OB resulted
in a signicantly higher larval mortality than treatment with either virus/botanical insecticide alone at the corresponding concentrations.
When consumed together (AZA and SpltNPV) larvae died signicantly faster compared with larvae consuming SpltNPV or AZA. These
results suggest that treatments with AZA and SpltNPV at appropriate combinations of concentration levels may result in improved control of tobacco cutworm compared with treatment with either AZA or virus alone.
 2006 Elsevier Inc. All rights reserved.
Keywords: Tobacco cutworm; Spodoptera; Nucleopolyhedroviruses; Azadirachtin; Biology; Development; Synergism; Mortality

1. Introduction
Environmental and human health concerns over excessive synthetic chemical insecticide use worldwide increasingly favor the development and marketing of alternative
and safer methods for pest control in many countries
(Cherry et al., 1997). Repeated exposure to chemical insecticides has given cause for concern for the health of farmers
(McConnell and Hruska, 1993; Tinoco and Halperin,
1998).The development of insecticide resistance coupled
with an increasing awareness of the possible detrimental
eects of intensive insecticide use has stimulated interest
in the development of integrated methods of pest control,
*

Corresponding author. Fax: +82 63 840 2118.

E-mail addresses: senthilkalaidr@hotmail.com,
(S.S. Nathan).

senthil@rda.go.kr

1049-9644/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2006.06.013

which reduce pesticide inputs and produce a more sustainable farming system (Carter, 1989). Insect viruses are
potential biological control agents of agricultural and forest pests. Most viral agents used to control pests are either
nucleopolyhedroviruses (NPV) (Mamestrin for Mamestra
brassicae Lin., Helicoverpa, Plutella and Labesa spp.) or
granuloviruses (GV) (Cp GV-Cyd-X for Cydia pomonella
Lin.) both of which belong to the family Baculoviridae.
Baculoviruses are rod-shaped double-stranded DNA genomes of about 88180 kilobases in size (Francki et al.,
1991). The nucleopolyhedroviruses (NPV) and granuloviruses (GV), make up the Baculoviridae. Both NPVs and
GVs are specic to the larval stage of their insect hosts.
The NPV have virions embedded in a crystalline matrix
of the protein polyhedrin. The virions vary in their conguration in that the nucleocapsids may be enveloped singly
or in multiples (Bonning and Hammock, 1996). The

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

occluded viruses are referred to as polyhedra. The granuloviruses (GV) have one, or rarely two, virions embedded
in a crystalline matrix of granulin. The nucleocapsid is
always enveloped singly (Bonning and Hammock, 1996).
A unique feature of baculoviruses is that they have
adapted both to ecient replication in the insect host and
environment outside by producing two morphotypes, budded virus (BV) and occluded virus (OV). In NPV infected
larvae, a critical level of viral infection and accumulation
of virions occurs, generally by 5 days. There are many
examples where fatal infection is slower (i.e. the gypsy
moth) also many factors play a role-age in insects, virus
concentration and temperature etc. In the case of GVs this
period is generally extended to 714 days. NPV-infected
larvae swell slightly and may become pale due to the accumulation of OVs, and often climb towards the upper foliage of the host plant where they die (i.e. Helicoverpa)
(Bilimoria, 1986; Inceoglu et al., 2001; Nakai et al.,
2002). Baculoviruses possess several features including narrow specicity, adequate pathogenicity, ease of genetic
manipulation, minimal residue problem, in vivo and in vitro
production capability, and compatibility with integrated
pest management programs make them suitable biological
agents for pest control (Inceoglu et al., 2001; Richards
et al., 1998). Baculoviruses, may provide an alternative to
chemical insecticides for control of insect pests due to their
advantageous characteristics including safety for non-target organisms and the environment (Kunimi et al., 1996;
Hunter-Fujita et al., 1998). Some NPVs like Spodoptera littoralis NPV, Lymantria dispar NPV and Anticarsia germmatalis NPV have successfully been used for the control
of insect pests in agriculture (Black et al., 1997; Moscardi,
1999; Inceoglu et al., 2001). Despite their advantages, there
are also disadvantages (Federici, 1993, 1997; Moscardi,
1999; Lacey et al., 2001). Baculoviruses are of potential
problems for widespread commercial use, including relatively slow speed of kill, narrow specicity, instability due
to ultraviolet light in eld, high production costs and short
shelf life compared with chemical pesticides. (Payne, 1988;
Cunningham, 1988; Inceoglu et al., 2001; Lavina et al.,
2001; Ishii et al., 2003). In addition, technical and economical diculties for in vitro commercial productions are
encountered (Moscardi, 1999).
This problem is a target for improvement by combination of other insecticides especially plant based insecticides
(Shapiro et al., 1994; Cook et al., 1996; Senthil Nathan and
Kalaivani, 2005; Senthil Nathan et al., 2005a).The tobacco
cutworm Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a destructive pest and has the potential to be a
serious pest of forage crops in Asia. S. litura Fabricius
(Lepidoptera: Noctuidae) is polyphagous and has about
150 host species including cotton, soybean, celery, tomato,
chrysanthemum and sunower (Rao et al., 1993; Gothama
et al., 1995). It is one of the most economically important
insect pests in many countries including India, Japan, China and other countries of Southeast Asia (Senthil Nathan
and Kalaivani, 2005; Senthil Nathan et al., 2005a). Many

97

strains of tobacco cutworm have developed resistance to

some widely used insecticides (i.e. endosulfan and chlorpyriphos) (Brewer and Trumble, 1989; Kranthi et al.,
2002). Farmers usually make several applications of synthetic insecticides for the control of this pest during the
growing season in countries like India (Kranthi et al.,
2002) and USA (Brewer and Trumble, 1989) including
Central America (Hruska and Gould, 1997).
The insecticidal, repellent and antifeedant properties of
neem derivatives have been known for three decades.
Although several potentially insecticidal compounds occur
in neem seed extract (Ishida et al., 1992), the principle
active ingredient in most formulations is the tetranortriterpene azadirachtin (Senthil Nathan et al., 2005b,c), AZA1
can be an eective insecticide at low concentrations against
some insect species (i.e. Locusta migratoria L.) (Sieber and
Rembold, 1983).
Combining insect pathogenic viruses with insecticides
for improved pest control is of considerable interest in pest
control because of decreasing time to kill the target pest
(Jaques and Morries, 1981; Murugan et al., 1999; Senthil
Nathan and Kalaivani, 2005). Earlier studies by Shapiro
et al. (1994), Cook et al. (1996) and Senthil Nathan and
Kalaivani (2005), reported enhancement of virus activity
by neem derivatives. Those results indicate AZA treatment
might have directly aected the insects gut lining to allow
easier pathogen penetration.
In continuation with our pervious work (Senthil Nathan
and Kalaivani, 2005), we studied the interactive eects of
AZA and SpltNPV on tobacco cutworm. This study was
undertaken to determine the nature of interaction between
virus and botanical insecticide when applied simultaneously and to evaluate the potential use of AZA and SpltNPV
combination for control of tobacco cutworm.
2. Materials and methods
2.1. Laboratory mass culture of S. litura
Spodoptera litura larvae were collected from castor (Ricinus communis L.: Euphorbiaceae) plantation elds in and
around Namakkal district, Tamil Nadu, India. Larvae were
reared in the laboratory on castor leaves. The castor plants
were grown in a eld and were 1.52 months old. For the leaf
tests and mass culture we used mature leaves (75125 cm2)
that were removed from the upper third of the plants as needed. Pre-pupae were separated and provided with vermiculture clay as pupation sites. Emerging adult moths were
transferred to cages and fed on a 10% sucrose solution fortied with a vitamin mixture (Multidec drops, Ashok pharmaceuticals, Chennai-24, India) to enhance oviposition. Moths
were transferred at a ratio of 1 male:2 females to oviposition
cages containing castor leaves and covered with sterilized

1
Abbreviations used: AZA, Azadirachtin; SpltNPV, Spodoptera nucleopolyhedrovirus; OB, occlusion bodies.

98

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

muslin cloth for egg laying. The muslin cloths containing

eggs were removed daily and eggs present were surface sterilized (to prevent virus infection) in situ by dipping in 10%
formaldehyde solution for 25 min, then washing with distilled water for 2 min. The muslin cloths containing eggs
were moistened and kept in plastic containers
(500 356 200 mm) to allow hatching. All experiments
and culture were carried out at 28 2 C, 65% relative
humidity, with a 14:10 light/dark cycle.
2.2. Preparation of NPV
Freshly emerged fourth instars of S. litura reared in the
laboratory were inoculated with a virus suspension (Spodavax (SpltNPV), T. Stanes & Company Ltd., Coimbatore,
India) containing 1 108 occlusion bodies (OB/ml). The
NPV formulations were applied to leaves with a regulator-controlled spray applicator, which were then fed to
the larvae, leading to infection and death of the larvae.
Virus-killed were thoroughly macerated in distilled water
and mixed by homogenizer. The viral suspension was ltered through a sterilized muslin cloth, and the ltrate
was centrifuged at 90g (rcf) for 5 min. The supernatant
was discarded and the sediment, containing the OB, was
diluted with distilled water. The concentration of the viral
suspension was determined by counting in a Neubauer
haemocytometer. Viral suspensions ranging from 102 to
108 OB/ml were prepared by diluting with distilled water,
and then stored in a deep freezer.

every 24 h and nal mortality was recorded after 12 days.

The percentage mortality was calculated by using the formula (1) and corrections for natural mortality when necessary were done by using Abbots (1925) formula.
Percentage of mortality

Number of dead larvae

 100
Number of larvae introduced

The percent mortality data after correction (Abbot,

1925) were subjected to probit analysis (Finney, 1971) to
calculate mean eective concentration (EC50). From the
mean eective concentration, the treatment doses were
selected for biological studies.
2.5. Developmental study

Azadirachtin (purity > 99%, received from Dr. M. Ishida, Central Research Laboratories, Taiyo Kagaku Co.
Ltd., Japan) was dissolved in isopropanol and dierent
concentrations were prepared by dilution with isopropanol.

Emerged second instar S. litura were taken from a laboratory culture. Larvae which had been reared on the fresh
castor leaves (75125 cm2)that had been treated with 0.1,
0.25 and 0.5 ppm of AZA and 102, 103 and 106 OB/ml of
NPV. The uneaten leaves were removed after 24 h, and
replaced with fresh untreated leaves. Control leaves were
treated with isopropanol (0.1%) and air-dried. A minimum
of 20 larvae/concentration were used for all the treatments
and these treatments were replicated ve times as like bioassay experiment. The treated and control groups were
maintained at 27 2 C in a 14:10 L:D and 85% RH.
Records were made daily of living and dead individuals.
The duration of larval and pupal stages were recorded.
Pupae of all test larvae were weighed on the rst day after
pupation. Deformities in the pupae or emerged adults were
recorded. Fecundity was determined by rearing the moths
emerging from the treated larvae with normal adults of
the opposite sex from the laboratory cultures by observing
number of progeny produced. Pairs were conned in sleeve
containers, which contained castor leaves for oviposition.
The longevity of the adults was also recorded.

2.4. Bioassays and treatments

2.6. Statistical analysis

Bioassays were performed with rst to fourth instars of

S. litura larvae using concentrations of 0.1, 0.25, 0.5 and
1 ppm of AZA and 102, 103 and 106 OB/ml of NPV. The
fresh castor leaves (75125 cm2) were sprayed with dierent
concentrations of azadirachtin (0.101 ppm) and SpltNPV
(102106 OB/ml) on both surfaces and air-dried. Control
leaves were treated with isopropanol (0.1%) and air-dried.
The formulations were applied to leaves with a regulatorcontrolled aerial spray applicator. Approximately 2 ml of
virus or AZA solution per leaf was applied on both leaf
surfaces. A minimum of 20 larvae/concentration were used
for all the treatments and these treatments were replicated
ve times (n = 100). The uneaten leaves were removed after
24 h, and replaced with fresh untreated leaves, whereby the
larvae were disturbed as little as possible. After leaves were
replaced, the test arenas were positioned vertically for few
minutes, with the side occupied by the larvae down, to
allow the larva to settle on the leaf. Mortality was recorded

Eective concentration was calculated by using Probit

analysis (Finney, 1971). Data from developmental study
and mortality were subjected to analysis of variance (analysis of variance of arcsine square root transformed percentages). Dierences between the treatments were determined
by Tukeys multiple range test. Dierence between means
were considered signicant at P 6 0.05 (Snedecor and
Cochran, 1989; SAS Institute, 2001).

2.3. Azadirachtin

3. Results
3.1. Eect of AZA and NPV on survival and mortality of
S. litura
Mortalities of rst and second instar larvae were higher
for combined virus and botanical insecticide treatment.
AZA and SpltNPV individual treatment required a longer
time to produce mortality but it was signicantly reduced

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

100

99

90

Mean larval mortality in percentage

80

70

ab

ab
Control

60

50

c
c

40

AZA- 0.25

bc

AZA- 0.50

30

AZA- 0.10

bc

NPV- 10 2

NPV- 10 3

20

d
10

d
d

d
d

NPV- 10

AZA-0.10 + NPV- 10 2

AZA-0.25 + NPV- 10 3

Larval instar

Fig. 1. Mean (SEM) percentage mortality of rst to fourth instar larvae of S. litura after treatment with AZA and SpltNPV. Means (SEM standard
error) followed by the same letters above bars indicate no signicant dierence (P 6 0.05) according to a Tukey test.

3.2. Eect of AZA and NPV on biology of S. litura

AZA and SpltNPV tested at all concentrations (individually and as combinations) aected larval performance and
extended the duration of the larval stage. Larval instar had
a negative relationship with susceptibility to S. litura (i.e.
younger instars were more susceptible than older instar
for NPV). The incorporation of the AZA and SpltNPV

A 0.9
AZA concentration in ppm

0.8
0. 7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
First instar

Second instar

Third instar

Fourth instar

First instar

Second instar

Third instar

Fourth instar

B 105
Virus concentration in OB (1x10)

in combined treatment in particular rst (F = 24.9051;

df = 3; P < 0.0001) and second (F = 24.9223; df = 3;
P < 0.0001) instar. Larval mortality was signicantly
increased by the combination of AZA and SpltNPV at
AZA-0.50 ppm + SpltNPV-106 over that in treatments
with either virus alone at the corresponding concentration
levels (Fig. 1). An interaction appeared to occur between
AZA and SpltNPV when consumed together on treated
castor leaves.
Concentration-dependent mortality was evident after
individual treatment with AZA and SpltNPV, and higher
mortality occurred in the combination treatment. The combined treatment of AZA and SpltNPV caused almost 100%
mortality at maximum concentration tested (Fig. 1).
Therefore, there appears to be a biologically signicant
interaction occurring between the azadirachtin and the
virus in determining the speed and overall mortality of
the larval population. Eective concentrations (EC50) are
shown in Fig. 2. For both AZA (Fig. 2A) and SpltNPV
(Fig. 2B), EC50 values were lowest in rst instar larvae.
AZA killed the fourth instar larvae within 14 days with
peak host mortality occurring 2 days after treatment
(Fig. 3A). SpltNPV killed the host as like AZA, with peak
mortality at mean of 3 days after treatment. Each combination treatment had a typical curve with two peaks, one
occurring 3 days after treatment and other occurring 5 days
after treatment (Fig. 3B).

10

10

10

10

Fig. 2. Eective concentrations (EC50) of Azadirachtin (A) and SpltNPV

(B) against rst to fourth instar larvae of S. litura (values are means of ve
replicates and standard error).

in the castor leaves diet signicantly inuenced the development of neonate S. litura larvae in a concentration and
combination-dependent manner (Figs. 46). AZA had sig-

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

14

Daily mean mortality in percentage

100

12

Control
AZA- 0.10
AZA- 0.25
AZA- 0.50

10

NPV- 10

NPV- 10 3

NPV- 10

6
4
2
0
1

5
6
Days after treatment

35

10

AZA-0.10 + NPV-10 2
AZA-0.25 + NPV-10

30

Daily mean mortality in percentage

Control
25

20

15

10

0
1

10

11

Days after treatment

Fig. 3. Daily mean (SEM) percentage of mortality of S. litura after individual (A) and combined (B) treatment with AZA and SpltNPV. Means of ve
replications with 20 larvae per replicate.

nicant antifeedant eects on the larvae, and these eects

were more pronounced in rst and second instars
(Fig. 4). There were no signicant dierences between the
individual concentration treatments, but signicant dierences were recorded between the combinations. The duration of the larval (F = 24.79; df = 3; P < 0.0001) and
pupal (F = 24.81; df = 3; P < 0.0001) (Fig. 4A) stage was
signicantly extended in the combined treatment of
0.25 ppm AZA + 103 OB/ml of NPV, compared to the
controls and individual treatments (Figs. 4A and B). Adult
male (F = 22.73; df = 3; P < 0.0001) and female
(F = 24.59; df = 3; P < 0.0001) longevity was reduced by
the combination treatments (Fig. 5). Fecundity was also
signicantly reduced in combined treatments compared to
the control and individual treatment (F = 24.93; df = 3;
P < 0.0001) (Fig. 6).
The combination of AZA and SpltNPV also decreased
the percentage of larvae that successfully moulted. For all

of the measured parameters (larval (F = 24.78; df = 4;

P < 0.0001) and pupal (F = 24.49; df = 4; P < 0.0001)
duration adult longevity (male-F = 14.66; df = 4;
P < 0.0001, female-F = 23.38; df = 4; P < 0.0001) except
number of eggs and mortality) there was no detectable difference in results among the three concentrations of AZA
tested.
Growth-retardation eects such as formation of larvalpupal intermediates and crippled wings with abdomens
occurred in some treatments. These eects were signicant
at higher concentrations of AZA. At higher concentrations
of AZA and SpltNPV slowed larval moulting and the larvae failed to moult.
4. Discussion
Environmental and health problems associated with
chemical-based insecticides have prompted the search for

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

101

40
35
Control
30

AZA-0.10

Days

25

AZA-0.25
AZA-0.50

20

SpltNPV-10
15
SpltNPV-10

2
3

10

SpltNPV-10

AZA-0.10+SpltNPV-10

AZA-0.25+SpltNPV-10

2
3

Mean larval duration in days

25
a
20
b

Control

AZA-0.10

c
15

Days

AZA-0.25

c
d

AZA-0.50

10

SpltNPV-10

SpltNPV-10

SpltNPV-10

AZA-0.10+SpltNPV-10
AZA-0.25+SpltNPV-10

2
3

Mean pupal duration in days

Fig. 4. Mean (SEM) total duration of larval (A) and pupal (B) period of S. litura after treatment with AZA and SpltNPV. Means ((SEM) standard
error) followed by the same letters above bars indicate no signicant dierence (P 6 0.05) according to a Tukey test.

12

Male

Female
a

10

a
a
a

Control

a
b

b
bc

b
bc

Days

AZA-0.1

AZA-0.25

AZA-0.5

SpltNPV-10 2
4

c
d

SpltNPV-10
SpltNPV-10

AZA-0.10+SpltNPV-10

AZA-0.25+SpltNPV-10

Mean adult longeivity in days

Fig. 5. Mean (SEM) total longevity of adult (male and female) S. litura after treatment with AZA and SpltNPV. Means ((SEM) standard error)
followed by the same letters above bars indicate no signicant dierence (P 6 0.05) according to a Tukey test.

102

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

AZA-0.25 + SpltNPV-10

AZA-0.10 + SpltNPV-10 2

SpltNPV-10 6
SpltNPV-10

SpltNPV-10 2

AZA-0.50

AZA-0.25

b
a

AZA-0.10

Control

100

200

300

400

500

600

700

800

900

1000

1100

1200

Mean number of eggs laid by the female

Fig. 6. Mean (SEM) number of eggs (fecundity) laid by female S. litura after treatment with AZA and SpltNPV. Means ((SEM) standard error)
followed by the same letters above bars indicate no signicant dierence (P 6 0.05) according to a Tukey test.

ecologically acceptable pesticides (Wood and Granados,

1991). The use of genetically engineered crop plants
expressing insecticidal toxins from Bacillus thuringiensis
Berliner (Bt endotoxin) has introduced a new dimension
on complexity to the problem and is further confusing producers and regulatory agencies throughout the world
(Inceoglu et al., 2001). Resistance against this family of
insecticidal proteins is more and more common (i.e. Plutella xylostella L.) especially with the extensive use of genetically modied plants expressing Bt toxins. In one case, the
use of Bt toxin against diamondback moth (DBM, P. xylostella) has been abandoned due to resistance (Roush, 1997;
Inceoglu et al., 2001). Recent problems with public acceptance of transgenic crop such as Bt corn for human consumption are also important consideration even Bt is far
more widely used than baculoviruses.
Viruses, particularly those belonging to the family Baculoviridae, are one of the most promising biological insecticides to date (Lavina et al., 2001). The results of the present
study have shown that SpltNPV and AZA combination
can provide control of S. litura larval populations, which
is comparable with, or superior to, that provided by a
botanical insecticide or virus alone. This observation conrms that previously reported by Cook et al. (1996). Cook
et al. (1996), using pure azadirachtin content and a
decrease in the concentration of virus required to obtain
50% larval mortality. Spodoptera NPV has been reported
to be more eective against rst and second instar larvae
than against older tobacco cutworm larvae because young
larvae are positively phototropic, very mobile and wander
before they start feeding on the host plant or diet (Smith
and Vlak, 1988; Gothama et al., 1995).
The eects of azadirachtin and SpltNPV on insect development and mortality observed in the present study can be
compared to eects observed in other insects (Shapiro

et al., 1994; Cook et al., 1996; Senthil Nathan and Kalaivani, 2005; Senthil Nathan et al., 2005a). Similar eects
are suggested by the decrease in life span of adult male
and female and increased larval and pupal duration
observed in the bioassay (Murugan et al., 1999).
The present nding also showed reduced development
rate during rst to fourth instar, with reduced growth in
treated and infected larvae, is in conrmation with earlier
ndings (Shapiro et al., 1994; Senthil Nathan and Kalaivani, 2005). It may be concluded from this study that
extended larval period is coupled with lower developmental
rate with increased mortality. A similar result was reported
by Shapiro et al. (1994) using aqueous neem extract in
combination with (Lymantria dispar L.) gypsy moth NPV.
The present study shows that applications of AZA and
SpltNPV in combination treatments resulted in synergistic
eects on mortality of tobacco cutworm, regardless of the
eective concentration used. These results are also consistent with other workers (Cook et al., 1996; Senthil Nathan
and Kalaivani, 2005). When combined with Spodavax
(SpltNPV), the azadirachtin increased the percentage of
larval mortality by 4050% when compared with treatments containing only the virus (Shapiro et al., 1994; Cook
et al., 1996). The decreased time to kill was reported by
Shapiro et al. (1994) and Cook et al. (1996), using whole
neem extract and pure azadirachtin content and a decrease
in the concentration of virus required to obtain 50% larval
mortality (Shapiro et al., 1994; Cook et al., 1996; Senthil
Nathan and Kalaivani, 2005), Enhancement of viral activity against lepidopteran pest insect larvae by adding various compounds like boric acid (Shapiro and Bell, 1982),
chitinase (Shapiro et al., 1987), optical brighteners (Shapiro and Robertson, 1992; Monobrullah, 2003; Martinez
et al., 2003) and neem components (Shapiro et al., 1994;
Cook et al., 1996; Senthil Nathan and Kalaivani, 2005),

S.S. Nathan, K. Kalaivani / Biological Control 39 (2006) 96104

Bacterial toxin like Bacillus thuringiensis (Granados et al.,

2001) and nematodes (Gothama et al., 1995) to viral formulations have already reported. The advantage of adding
these compounds to Spodoptera NPV formulations is the
potential decrease in the pathogen dosage required to kill
larvae. The greatest advantage is the faster kill and negative eects on growth and development which could result
in lesser feeding damage to the host plant. Azadirachtin
treatment might have directly aected the insects gut lining
to allow easier pathogen penetration (Cook et al., 1996).
The combination of virus and neem derivatives confers
an advantage for pest management especially of a polyphagous pest like S. litura.
Azadirachtin has been shown to be the most active compound in the neem derivatives in terms of its eects on
insect physiology (Senthil Nathan et al., 2005b,c). This
activity is apparently strongest during pupation; pupae
were very susceptible following larva exposure (Smirle
et al., 1996). The chemical structures of the assayed compounds are with furan ring and an a, b-unsaturated ketone
in their A-ring (Ishida et al., 1992).
From the study it is concluded that azadirachtin potentiates the eect of SpltNPV by producing mortality with
in a short time and negatively inuences the larval growth
and development. The issue addressed in the present study
is the potential of applying the azadirachtin to the S. litura
virus bioinsecticide. From a cost standpoint, we argue
that using botanical insecticide in insect virus formulations will probably only be feasible if the concentration
of virus in spray applications can be signicantly reduced,
and/or if the AZA shows high synergistic activity in the
eld at low concentrations, and/or if the volume of water
applied in spray application of the virus can be reduced
yet remain eective in delivering it to the feeding site of
the pest (Martinez et al., 2000). In conclusion, AZA kills
a proportion of test larvae, and the survivors are then
infected and killed by NPV also AZA weakens a larvas
resistance to NPV. Formulation of virus with AZA may
appear to oer a valuable means of improving the ecacy
of the NPV.
Acknowledgments
The authors thank Mr. Karthikeyan, research assistant,
for his support during the research, M. Somasundram; M.
Palanivel for their eld assistance; K. Manavalasundram
for his technical assistance and thanks also to senior
authors graduate students for their voluntary help during
the research period. We thank Dr. M. Ishida, Central Research Laboratories, Taiyo Kagaku Co. Ltd., Japan for
providing neem components. Special thanks are also given
to two anonymous reviewers for their valuable comments
on an earlier draft of the manuscript. Financial help to
the rst author from the Rural Development Administration, NICS to conclude this work is gratefully
acknowledged.

103

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