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Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
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Pesticide Biochemistry and Physiology
journal homepage: www.elsevier.com/locate/pest
Physiological effect of chitinase purified from Bacillus subtilis against the
tobacco cutworm Spodoptera litura Fab.
Rajamanickam Chandrasekaran 1, Kannan Revathi 1, Selvamathiazhagan Nisha,
Suyambulingam Arunachalam Kirubakaran, Subbiah Sathish-Narayanan, Sengottayan Senthil-Nathan ⇑
Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Center for Excellence in Environmental Sciences (SPKCES), Manonmaniam Sundaranar University,
Alwarkurichi 627 412, Tirunelveli, Tamil Nadu, India
a r t i c l e
i n f o
Article history:
Received 5 January 2012
Accepted 2 July 2012
Available online xxxx
Keywords:
Bacillus
Chitinase
Enzyme
Molecular masses
Cutworm
Dietary utilization
Mortality
a b s t r a c t
An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC50) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed
the highest insecticidal activity at 6 lM concentration within 48 h. The nutritional indices were also
significantly affected by the 6 lM concentration (P < 0.05). Food consumption, efficiency of conversion
of ingested and digested food, relative growth rate, and consumption values declined significantly while
approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the
chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an
effective biocide and alternative to Bt toxin.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Pesticides are applied in agricultural systems for protecting
crops from damage by insects and disease. The use of pesticides
has also resulted in significant benefits effects to public health
and the environment. In general the amount of pesticides released
into the environment has risen significantly in the last five decades
[1,2]. However scientists constantly seek for an effective and
environmentally friendly method of controlling pests and diseases
[3–5].
Control of crop pests by the use of biological agents holds great
promise as an alternative to the use of chemicals. Secondary
metabolites and crude enzyme from microorganisms have been
used to control crop pest population [6].
Chitin is a long, unbranched polysaccharide of an amino sugar
N-acetyl-b-D-glucosamine linked together by b-1,4-glycosidic
linkages [7]. It is abundant in nature as a structural compound in
cuticle and integument of animals, especially in insects [8]. Chitin
is metabolized by various chitinases that are found in insects, bacteria, fungi and higher plants [9]. Chitinases belong to glycosyl
hydrolase families 18 and 19 according to the classification made
⇑ Corresponding author. Fax: +91 4634 283270.
E-mail
addresses:
[email protected],
(S. Senthil-Nathan).
1
These authors contributed equally to this work.
[email protected]
by Henrissat and Bairoch [10]. The family 18 chitinases have been
determined to possess a common (a/b) 8-barrel domain, comprising of eight a-helices and eight b-strands. The catalytic reaction of
the family 18 enzymes takes place through a retaining mechanism,
in which b-anomer is generated by hydrolysis of b-l,4-glycosidic
linkages. The chitinases of the family 19 have been described as
being similar to lysozyme and chitosanase in its mode of action
[11].
Insect growth and development are strongly dependent on the
construction and remodeling of chitinous structures [12]. Chitinase
induced damage to the peritrophic membrane in the insect gut
causes a significant reduction in nutrient utilization and consequently in insect growth [13]. Due to this, chitinase present in
the insect diet can decrease insect growth [14,15]. Chitinolytic
microorganisms have many potential applications as biocontrol
agents [16]. Over-expression of a chitinase in an entomopathogenic organism can increase insect mortality [17].
Chitinase plays a vital role in the control of plant pathogenic
fungi and raising plant disease tolerance. In the microbial degradation of chitin using chitinase, chitooligosaccahrides are produced,
which are further degraded to N-acetylglucosamine by chitobiase
[18]. Consequently, microbial chitinases have been isolated for
the production of chitooligosaccharides [19].
Bacillus species are well-known as producers of extra-cellular
enzymes, including cellulase, glucanase, amylase, proteinase and
chitinase, secondary metabolic products, and they are used
0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pestbp.2012.07.002
Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002
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R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
frequently in industrial fermentation [20,21]. Bacillus thuringensis
is notable as a source of insect toxins [9]. Bacillus subtilis (B. subtilis), is a gram-positive, non-pathogenic, spore forming bacterium
with notable chitinase activity [22].
Insects systematically synthesize and degrade chitin in a highly
controlled manner to allow ecdysis and regeneration of the peritrophic matrices. Some of the chemical compounds that disrupt
chitin metabolism, such as diflubenzuron, have been of special
interest for the control of agricultural pests [12].
Spodoptera litura Fab. (Lepidoptera: Noctuidae) is a polyphagous
insect, and worldwide it infests crops like cotton, groundnut, crucifers, tomato, tobacco, potato and soybean [23,24]. S. litura has
150 host species [25]. S. litura causes serious damage during early
seedling stage [25]. Farmers usually make several applications of
synthetic insecticides for the control of this pest during the growing season in India [26].
We have purified a B. subtilis isolate which secretes to a large
amount of extracellular chitinase shows high levels of pathogenicity against S. litura. In the present study, a chitinase was purified
and characterized from B. subtilis, and tested against S. litura.
2. Materials and methods
2.1. Isolation and screening of chitinase producing microorganism
Bacterial isolates were obtained from the soil in and around
Namakkal (11°7333300 latitude and 78°0666700 longitude) and
Tirunelveli (08°800 and 09°2300 latitude and 77°0900 and 77°5400 longitude) district, Tamil Nadu, India. From 25 soil samples (Table 1),
we isolated chitinolytic bacteria, which produced a zone of clearance in chitin media. The clear zones occur due to the hydrolysis
of colloidal chitin from the medium. We selected two chitinase
producing organisms (B. subtilis) for the purification of chitinase.
The soil organisms were screened on colloidal chitin agar plates
containing (g/l): chitin, 5.0; yeast extract, 0.5; (NH4)2SO4, 1.0;
Table 1
Isolation of chitinolytic bacteria from soils from Namakkal and Triunelveli district in
of Tamil Nadu, India.
S. No.
Soil samples collected
Chitinase
producing bacteria
Presence of zone
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Namakkal
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Triunelveli
Bacillus sphericus
Bacillus thuringiensis
Bacillus sphericus
Bacillus popilliae
Streptomyces
Bacillus subtilis
Bacillus megaterium
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus cereus
Bacillus cereus
Streptomyces
Pseudomonas fluroscence
Pseudomonas fluroscence
Bacillus subtilis
Baciillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus thuringiensis
Bacillus cereus
Bacillus cereus
Bacillus subtilis
Bacillus subtilis
Bacillus sphericus
Bacillus thuringiensis
+
+
+
+
+
+
+
+
+
+
+
+++
+++
+
+
+
+
+
+
+
+, Presence of zone in chitin agar plate; , represents absence of zone in chitin agar
plate, +++, used for chitinase enzyme purification.
MgSO47H2O, 0.3; and KH2PO4, 1.36, agar 20 pH 7.2 at 30 °C for
72 h [22]. Well separated colonies that showed the clear zone in
the chitin agar plates were selected. The identified cultures were
transferred into pure agar slants and the morphological, and the
physiological characters of microbes were analyzed according to
the Bergey’s manual determinative bacteriology [27]. Colonies that
showed a large zone of clearance were selected and used for
chitinase production.
2.2. Insect rearing
S. litura pupal cultures were collected from Project Directorate
of Biological Control, Bangalore, Karnataka, India. Emerging adult
moths were transferred to cages and fed on a 10% sucrose solution
to enhance oviposition. Moths were transferred at a ratio of one
male: two females to oviposition cages containing castor plant, Ricinus communis L. (Euphorbiaceae) leaves and covered with sterilized
muslin cloth for egg laying. The muslin cloths containing eggs were
removed daily and eggs present were surface-sterilized in situ by
dipping in 10% formaldehyde solution for 2–5 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. Larvae were reared in the laboratory
on R. communis leaves. All experiments and cultures were carried
out at 28 ± 2 °C, 65% relative humidity, with a 14:10 light/dark
cycle.
2.3. Preparation of colloidal chitin
Colloidal chitin was modified from the method of Lee et al. [28].
Twenty grams of chitin powder (RM1356-500G, Hi-media, India)
was added slowly into 350 ml of concentrated HCl and left at
4 °C overnight with vigorous stirring. The mixture was added to
2 L of ice-cold 95% ethanol with rapid stirring and kept overnight
at 35 °C. The precipitate was collected by centrifugation (EltekRC4100F) at 5000g for 20 min at 4 °C. The precipitate was washed
with sterile distilled water until the colloidal chitin became neutral
(pH 7.0).
2.4. Crude enzyme preparation
The chitinase producing B. subtilis strains were isolated from the
soil. These isolates were preserved in the colloidal chitin medium.
For the crude enzyme preparation, the isolates were grown in the
medium containing chitin, colloidal chitin-2 g/L; K2HPO4-1 g/L;
NaCl-5 g/L: MgSO47H2O-0.04 g/L; CaCl2-0.02 g/L [29] and cultured
in the 100 ml medium in 500-ml conical flasks and incubated
under shaking condition for about 8 days. The cultured fluids were
centrifuged at 10,000g for 20 min. The supernatant was allowed for
ammonium sulphate precipitation, allowed to stand overnight. The
pellet was centrifuged at 10,000g for 20 min. Then the precipitates
were dissolved in a small amount of 20 mM citrate phosphate buffer (pH 7.8) and extensively dialyzed against the same buffer. The
dialysate was used for further purification. Dialysates were subjected to ion-exchange chromatography on DEAE-Cellulose. The
peak fractions from this column containing the purified enzymes
were confirmed to contain 48 kDa chitinases by SDS–PAGE analysis, along with standard molecular weight markers. The recovery
of the yield and specific activity at various steps of the purification
procedure were summarized in the Table 2.
2.5. Enzyme purification
All the purification steps were carried out at 4 °C. The dialysate
sample was loaded directly into the DEAE-Cellulose column equilibrated with 20 mM citrate phosphate buffer (pH 7.5). The enzyme
Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002
R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
3
2.9. Food utilization, consumption and nutritional indices
Table 2
Purification of Chitinase from B. subtilis.
S. No.
Steps
involved
Total
protein (mg)
Total
activity (U)
Specific
activity (U/mg)
1
2
3
Culture supernatant
Dialysate
DEAE-cellulose
525
230
60
2954
2785
1150
6.52
11.2
42
1U = 1 lmol of reducing sugar per hour.
was eluted with a linear gradient from the citrate phosphate buffer
and eluted using the same buffer at a flow rate of 1 ml/h. One milliliter fractions were collected and assayed for chitinolytic activity.
2.6. Determination of molecular masses
SDS–PAGE was performed on 12% gels according to Lemmli
[30]. A standard protein ladder was used as a protein standard
marker. The molecular masses were also determined by chromatography (The same column as under the condition for the purification of the enzymes) using a standard curve obtained from
proteins with known molecular weights. The standard proteins
were BSA and cytochrome C.
2.7. Enzyme assay
Chitinase activity was assayed, based on dinitrosalicylic acid
(DNS) method [31]. The reaction mixture contained 70 ll of
enzyme solution and 70 ll of 1% colloidal chitin in 0.1 M citrate
phosphate buffer (pH 5.0). After incubation at 50 °C, 120 rpm for
30 min the mixture was heated in a boiling-water bath for 5 min
and then centrifuged at 3000 rpm for 5 min to remove extra chitin.
An equal aliquot vial containing 90 ll of the supernatant and equal
volume of DNS solution were mixed and heated in water bath for
15 min simultaneously, 30 ll of potassium sodium tartarate solution (400 g/L) was added. After cooling at 4 °C to room temperature, the absorbance at 540 nm (Systronic106) was measured.
The activity was calculated from a standard curve obtained using
known concentration of N-acetylglucosamine [32]. One enzyme
unit was defined as the amount of enzymes that produces 1 lmol
of reducing sugar per hour under reaction conditions. The protein
estimation was done by using Lowry et al. [33].
The S. litura third-instar larvae were starved for about 3 h, before that the initial weight of the larvae was measured. The larvae
were (10 larvae per concentration) allowed to feed on weighed leaf
treated with chitinase. After 24 h the remaining uneaten leaves in
the container were measured and replaced with fresh leaves daily.
Food consumption, weight gain and fecal weights were measured
using an electronic balance (Shimadzu, Japan). Each larva was
weighed, oven-dried and re-weighed to determine a percentage
dry weight of the experimental larvae after 24 h. The food ingestion was estimated by subtracting the diet remaining at the end
of the experiment from the total dry weight of the leaves. Fecal pellets were collected, weighed and then oven dried, and reweighed
to estimate the dry weight of the fecal material. Differences in
average weight of the larvae recorded at the beginning and at
the end of the period gave the gain in body weight while the mean
larval body weight gain was calculated using the formula: mean
weight = final weight initial weight [36].
The evaluations of nutritional indices of S. litura were done
according to Waldbauer [36]. Relative consumption rate RCR = dry
weight of food eaten/duration of feeding (days) mean dry weight
of the larva during the feeding period, the relative growth rate
RGR = dry weight gain of the larva during the period/duration of
feeding (days) mean dry weight of the larva during the feeding
period, approximate digestibility AD = 100 (dry weight of food
eaten dry weight of feces produced)/dry weight of food eaten,
Efficiency of conversion of ingested food ECI = 100 dry weight
gain of larva/dry weight of food eaten, and efficiency of conversion
of digested food ECD = 100 dry weight gain of the larva/(dry
weight of food eaten dry weight of feces produced). Larval
growth and food utilization were calculated after 24 h.
2.10. Larval weight versus larval duration
Emerged second instar larvae of S. litura were taken from a laboratory culture. Larvae which have been reared on the fresh castor
leaves that had been treated with different concentrations of chitinase (0.5, 1, and 2 lM). The uneaten leaves were removed after
24 h, and replaced with fresh leaves. Control leaves were treated
with distilled water and air-dried. A treated and control groups
were maintained at 28 ± 2 °C, 65% relative humidity, with a
14:10 light/dark cycle. Records were made daily of weight of living
and dead individuals.
2.8. Insect bioassay
2.11. Data analysis
Bioassays were performed with first, second- and third-instars
S. litura larvae using whole castor leaves amended with 2, 4 and
6 lM of chitinase. Control leaves received distilled water only. A
group of 10 larvae per concentration was used for all the treatments with five replicates. The uneaten diets were removed after
24 h and replaced with fresh untreated diets for control and
treated diets for treatments respectively. Deformities in the larvae,
pupae and emerged adults were recorded. Mortality was recorded
every 24 h, and final mortality was recorded after 12 days. The percentage mortality was calculated by using the formula (1).
Data from mortality and nutritional indices were expressed as
the mean of five replications and normalized by arcsine-square
root transformation of percentages. The transformed percentages
were subjected to analysis of variance (ANOVA) and were fitted
with linear regression using MinitabÒ16 software package. Differences between the treatments were determined using the
Tukey–Kramer HSD test (P 6 0.05) via the MinitabÒ16 software
package. The lethal concentrations LC50 was calculated using probit
analysis [35].
Percentage of mortality ¼
Number of dead larvae
100
Number of larvae introduced
ð1Þ
The percent mortality data after corrections [34] were subjected
to probit analysis [35] to calculate the mean of lethal concentration
(LC50). From the mean lethal concentration, the effective doses (EC)
were selected for biological studies.
3. Results
3.1. Molecular masses
The molecular weight of the protein eluted from the DEAE column was determined to be 48 kDa by SDS–PAGE. SDS–PAGE analysis of the purified enzyme revealed the presence of only one
protein band (chitinase) compared with a standard marker 116,
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R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
Fig. 1. SDS–PAGE of purified chitinase and standard marker proteins (116, 66, 45,
29 and 20 kDa). After electrophoresis, the gel was stained with Commassie blue. L1
and L2 lanes are pooled material of B. subtilis and L3 and L4 are peak material from
the DEAE column.
Fig. 2. Effects of chitinase on S. litura first (1), second (2) and third instar (3) larva
(6 lM) [C-Control, T-treated].
66, 45, 29 and 20 kDa. The molecular weight of the chitinase isolated from the bacteria was 48 kDa (Fig. 1).
100
a
I instar
II instar
a
III instar
The results showed that the purified enzyme hydrolyzed colloidal chitin. Our result indicates the presence of chitinases in purified enzyme preparation. The pure chitin prepared from colloidal
was used for the experiment. This hydrolysis of colloidal chitin
has showed the activity. During hydrolysis the enzyme released
reducing sugars from the colloidal chitin.
Percentage of mortality
a
3.2. Substrate specificity
80
c
3.4. Food utilization, consumption and nutritional indices
Chitinase treatment greatly reduced the relative consumption
rate and relative growth rate of the larval stage of S. litura. The
growth reduction was dependent on the concentration of chitinase.
The larval growth was at maximal level in control larvae. The chitinase containing diets altered growth parameters; the growth rate
is affected more than the relative consumption rate (Table 3). After
treatment with chitinase, low level of food consumption was resulted with less growth rate.
Approximate digestibility of treated larvae was gradually increased. The efficiency of food conservation of chitinase treated
larvae was significantly lower than control larvae. Significant
reduction of all nutritional indices was observed (F = 49.91;
df = 3.69; P = 0.001) in 2, 3 and 4 lM concentrations. Regression
between the relative growth rate and the relative consumption
c
c
20
d
d
0
Cont 2uM 4uM 6uM
The peak fractions from the DEAE column were used for application. Chitinase-treated leaves significantly reduced the growth of
the larvae. Mortality rate was gradually increased in first, second
and third instar due to the treatment of chitinase. Control larval
mortality was reached maximum of 3.8% (±0.6) in third instar. In
all the larval instar, treatment with chitinase caused mortality
and the percentage of mortality decreased slightly but not significantly in third instar.
The lowest (2 lM) concentration of the chitinase produced the
lowest mortality in third instar larvae, but it caused several abnormalities in larvae (Fig. 2). We noted that the treated larvae were
physiologically affected and malformed in larval to adult stage
when compared to control. At concentrations of 4 lM and 6 lM,
larval mortality were significantly different from the control in
the first (F = 49.12; df = 3.11; P = 0.001), second (F = 40.43;
df = 3.11; P = 0001) and third instar larvae of S. litura (F = 43.18;
df = 3.11; P = 0.001) (Fig. 3).
b
40
d
3.3. Mortality and growth of S. liutra after treatment with chitinase
b
b
60
Cont 2uM 4uM 6uM
Cont
2uM 4uM 6uM
Fig. 3. Percentage mortality of first, second and third instar of S. litura after
treatment with chitinase (Con-Control, 2, 4 and 6 lM). Means (SEM±) followed by
the same letters above bars indicate no significant difference (P < 0.05) according to
a Tukey test.
rate were plotted to determine the effects of chitinase against S.
litura and the regression of RCR–RGR for control (r2 = 0.897), and
the chitinase treated (r2 = 0.953) were significantly different in
treated compared to control. The regression lines represent the reduced growth level of the larvae which fed on the chitinase treated
leaves (Fig. 4).
3.5. Larval weight versus larval duration
Compared to the control larvae weight, chitinase exposed larvae
lost body weight. At high concentration it produced mortality and
also larvae did not survive as long as the controls. The third instar
larva fed with chitinase treated leaves at different concentrations
(0.5, 1 and 2 lM) for 1 h and then they were allowed to feed on untreated leaves, and several effects were noted. Larvae lost weight
and consumed significantly less diet than those exposed to the
lowest concentration (0.5 lM) and controls (Fig. 5). Survival periods of the larvae were calculated up to third to fourth instar and
the survival of larvae when treated with chitinase (1 and 2 lM)
were significantly different (F = 8.56, df = 3.36; P = 0.0001). The
longest survival periods were obtained in the control.
4. Discussion
Chitinase has been used previously as an insecticide and a fungicide [3]. Chitinase disrupts the peritrophic membrane and enables microorganisms and pathogens to invade the hemocoel
[37]. Bacteria produce chitinase for the use of chitin as nitrogen
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R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
Table 3
Nutritional indices of third instar larvae of S. litura after treatment with chitinase.
Chitinase (lM)
S. No.
1
2
3
4
RGR (mg/mg/day)
RCR (mg/mg/day)
b
2
3
4
Control
b
0.045 ± 0.0021
0.040 ± 0.0010c
0.022 ± 0.0006d
0.066 ± 0.0010a
0.94 ± 0.0057
0.87 ± 0.0058c
0.73 ± 0.0115d
1.04 ± 0.0051a
ECI (%)
ECD (%)
b
25.28 ± 0.85
20.96 ± 0.59c
18.22 ± 0.51d
30.42 ± 1.10a
AD (%)
b
32.700 ± 0.72
24.600 ± 0.74c
19.680 ± 0.73d
38.740 ± 0.65a
44.36 ± 0.25c
46.26 ± 0.39b
47.60 ± 0.79a
41.48 ± 0.57d
Means (±SE) followed by the same letters within columns of indicate no significant difference (P < 0.05) in a Tukey test.
0.09
µ
0.08
2
Relative growth rate (mg/mg/day)
y = 0.0738x - 0.00123; R=0.897
0.07
0.06
0.05
0.04
0.03
0.02
2
0.01
y = 0.0505x-0.00272; R=0.953
0.00
0.0
0.2
0.4
0.6
0.8
1.0
Relative consumption rate (mg/mg/day)
Fig. 4. Regression equation and correlation between relative consumption rate and
relative growth rate of S. litura fed on leaves containing chitinase with different
concentration and larvae with different quantities of control leaves.
500
Control
0.5 µM
1 µM
2 µM
Larval weight in mg
400
300
200
100
0
2
4
6
8
10
12
14
16
Number of days after treatment
Fig. 5. Larval weight of S. litura after treatment with chitinase (0.5, 1 and 2 lM)
(values are means of five replicates).
and carbon sources [38]. Most of the chitinases used as insecticides
are purified from the bacteria like, Bacillus [39,40], Pseudomonas
[41], and Streptomyces spp. [42].
Bacillus thuringiensis also produce several chitinases with different molecular weights: 66, 60, 47 and 32 kDa [40]. This type of the
chitinolytic enzyme is used as an antagonist and moreover, used as
a defense mechanism against chitin containing insects [43].
Bacterial chitinolytic enzymes have been used to enhance the
activity of microbial insecticides including Bacillus thuringiensis
and a baculovirus. Larvae of spruce budworm, Choristoneura fumiferana, died more quickly when exposed to a chitinase-Bacillus mixture than when exposed to the enzyme or bacterium alone [44].
The chitinase was caused perforations in the gut peritrophic membrane, facilitating entry of the pathogens into the hemocoel of susceptible insects [45].
The degradation of chitin requires more than one enzyme type.
Endochitinases produce chitololigomers converted into monomers
by exochitinase N-acetylglucosaminidases and then later the
enzyme cleaves N-acetylglucosamine from non-reducing ends. It
favors smaller substrates than chitinases [46,47]. Chitinolytic enzymes associated with the ecdysial cycle have been established
to act synergistically in cuticular chitin degradation [48].
In a previous study [49], chitinase was purified from the Bacillus
spp, and has been analyzed as insecticides. Previously chitinase has
been combined with an insecticidal protein, Bt toxin against S.
litura neonate larvae [50]. Also it was shown that the crude chitinase from B. circulans could increase the efficacy of B. thuringiensis
spp. kurstaki against diamondback moth larvae [51]. Chitinase was
used as insecticide in bioassays that decreased the LC50 value of
crystal protein produced by Bacillus thuringiensis against S. exigua
and H. armigera [52]. Apparently, chitinases found in the gut have
the ability to decrease digestive function in addition to their role in
breaking down the chitin present in the gut lining or peritrophic
membrane [9].
An enhanced toxic effect towards S. littoralis also resulted when
a combination of low concentrations of a truncated recombinant Bt
toxin and a bacterial endochitinase were incorporated into a semisynthetic insect diet [50]. Crude chitinase preparations from B. circulans enhanced the toxicity of Bt kurstaki towards diamondback
moth larvae [53].
The chitinase tested in our study was similar to that reported by
Smirnoff and Valero [51], Sneh et al. [54,55] and Wiwat et al. [53].
Microbial chitinases may partially digest the peritrophic membrane, assisting the microbes and their toxins in penetration of
the peritrophic membrane [54]. Compared to the above results
our present study with B. subtilis, chitinase has more effectively
controlled S. litura.
The relative growth rate and the relative consumption rate (RGR
and RCR) of Spodoptera strains were decreased significantly when
treated with toxins and secondary metabolites identified from
Bacillus [56]. Further the growth rate of third instar Spodoptera
strains were reduced, and the developmental periods were extended in the treated secondary metabolites from Bacillus
[57,58]. Decreased larval growth was coupled with lower RGR,
because the food was retained in the gut of the larvae for maximum of approximate digestibility to increase the nutrition.
The regression coefficients of the RCR–RGR relations for control
and treated animals were significantly different; showing that the
reduction in growth of larvae fed on chitinase containing leaves
was not entirely a result of lower food intake. The chitinase
reduced RGR and RCR with significant change in the ECI.
5. Conclusion
Bacterial chitinases emerge with properties that make them
exclusively useful for pest control. Our present study reveals that
purified chitinase from B. subtilis is effective against the S. litura
Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002
6
R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx
through increased larval mortality and reduced larval weight and
growth. This may serve as an effective biopesticide to control
tobacco cutworm compared to Bt and other toxins. Also it is a less
expensive and more accessible one. We are continuing to develop
bacterial chitinase as a biocide for insects and plant pathogens, by
introducing its gene into rice plant.
[23]
[24]
Acknowledgments
[25]
This research was supported by the Department of Science and
Technology (DST), Government of India (SR/FT/LS-161/2010 –
under science and engineering board). The authors sincerely thank
to Dr. Richard W. Mankin, USDA-ARS for his suggestion and comments. Technical assistant from Mr. K. Karthikeyan is gratefully
acknowledged. We are grateful to three anonymous reviewers for
comments on an earlier version of the manuscript.
[26]
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