isolation and characterization of native bacillus...
TRANSCRIPT
H. AMMOUNEH, M. HARBA, E. IDRIS, H. MAKEE
421
Turk J Agric For
35 (2011) 421-431
© TÜBİTAK
doi:10.3906/tar-1007-1117
Isolation and characterization of native Bacillus thuringiensis
isolates from Syrian soil and testing of their insecticidal
activities against some insect pests
Hassan AMMOUNEH*, Muhanad HARBA, Emad IDRIS, Hayat MAKEE
Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria,
Damascus, P.O. BOX 6091 - SYRIA
Received: 27.07.2010
Abstract: Bacillus thuringiensis was detected in 12.5% of soil samples collected from diff erent regions in Syria and 25 B.
thuringiensis isolates were found to be highly toxic to larvae of Ephestia kuehniella Zeller, Phthorimaea operculella Zeller,
and Cydia pomonella L. (Lepidoptera), but not to the larvae of Culex quinquefasciatus (Diptera). Light microscopy
investigation showed the presence of bipyramidal and cuboidal parasporal bodies produced by these isolates. Th e
existence of diff erent cry genes in the tested isolates was studied using a PCR strategy with a set of general primers
recognizing some of the cry genes reported in the relevant literature. Primers corresponding to 2 types of cry genes (cry1
and cry2) successfully amplifi ed DNA in all of the tested isolates. Moreover, the proteins encoded by these genes were
detected in the SDS-PAGE of the purifi ed parasporal bodies. Th e 50% lethal concentration of the spore-crystal mixture
of the 25 isolates against E. kuehniella larvae varied from 8.4 to 97.6 μg g-1. A comparison of the LC50
values of the tested
isolates with those of the reference strains B. thuringiensis kurstaki HD-1 and HD-73 (20.8 and 46 μg g-1, respectively)
showed that some of these isolates have a higher toxicity potential. Moreover, fl agellar serotyping revealed that 4
isolates, which were among the most toxic, belonged to serotype kurstaki. Th is study constitutes the fi rst isolation and
characterization of local B. thuringiensis isolates in Syria. Some of these isolates exhibit toxic potential and, therefore,
could be adopted for future applications to control some important insect pests.
Key words: Bacillus thuringiensis, Ephestia kuehniella, soil, Syria
Research Article
* E-mail: ascientifi [email protected]
Introduction
Th e greatest successes in microbial pesticides
have come from the use of commercial preparations
of Bacillus thuringiensis (Bt). Th ese have been the
most successful biological pest control products
worldwide, and 95% of microbial pesticides sold are
of this bacterial agent, with annual sales estimated
at $100 million (Kaur 2002; Federici et al. 2006). Bt
is a rod-shaped, gram-positive bacteria that occurs
naturally in soil, dead insects, water, and grain
dust (Lambert and Peferoen 1992). During the
sporulation process, these bacteria produce large
crystal proteins that are toxic against many insect
pests. When orally ingested by insects, this crystal
protein is solubilized in the midgut, forming proteins
called delta-endotoxins. Th e toxicity of these crystals
Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against
some insect pests
422
to the insects is determined by the presence of the
specifi c receptors in the midgut epithelium (Bravo
et al. 2007). Because delta-endotoxins are generally
safe to vertebrates and benefi cial arthropods and
are oft en highly toxic to insect pests at relatively low
doses, genes encoding these proteins (cry genes) were
among the fi rst to be used in genetic engineering
of plants for enhanced insect resistance (Roh et al.
2007).
Th e cry gene content of Bt strains is known to be
related to the toxicity (Federici et al. 2006), and the
identifi cation of cry genes by means of polymerase
chain reaction (PCR) has been exploited to predict
insecticidal activities of the strains and to determine
the distribution of cry genes within a collection
of Bt strains. PCR is progressing as a rapid tool
for preliminary characterization of Bt, but since
the primers are designed against known genes, the
technique presents limitations in the search for novel
cry genes. Moreover, the reliability of insecticidal
activity prediction based on PCR results is dependent
on the expression of the genes. Genes lacking
functional promoters or genes that are weakly
expressed will produce an erroneous prediction.
A thorough characterization of Bt strains should
therefore be achieved by other means. Consequently,
determination of parasporal crystal composition and
toxicity by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and bioassays is a
critical complement to gene identifi cation (Porcar
and Juárez-Pérez 2003).
For the last 3 decades there has been an intense
interest in collecting and analyzing novel Bt strains
derived from various environmental samples.
However, Syria corresponds to an area of the world
not explored before in the search for Bt isolates.
In this study, Bt isolates were collected from soil
samples distributed throughout the interior of Syria.
A complex approach was used in order to both
characterize the isolates and to assess their potential
as an active ingredient in a local bioinsecticide. Th e
analyses performed included light microscopy, testing
of the insecticidal activity against some insect pests
(especially Ephestia kuehniella), and determination
of cry gene content by PCR and SDS-PAGE analysis
of the parasporal crystal proteins.
Materials and methods
Sample collection
Samples were collected from soils from diff erent
regions distributed widely between 33.40°N and
37.17°N and 35.40°E and 42.30°E in Syria (Figure 1).
Th e sampling sites had not been previously sprayed
with Bt-based microbial insecticides. We attempted
to collect soil from locations that were as diverse
as possible. Soil samples were collected by scraping
off surface material with a sterile spatula and then
obtaining approximately 100-g samples from 2-5 cm
below the surface. Samples were then stored at 4 °C
until use.
Isolation of spore-forming Bacillus isolates
Th e acetate selection protocol of Travers et al.
(1987) was used with some modifi cations to obtain
spore-forming Bacillus isolates from soil. To 10 mL
of Luria-Bertani broth, 1 g from each soil sample
was added (Maniatis et al. 1989) and buff ered with
sodium acetate (0.25 M, pH 6.8) in a 125-mL fl ask.
Th e broth was incubated in a shaker at 200 rpm for
4 h at 30 °C. A 1-mL aliquot was heated to 80 °C for
15 min in a thermomixer (Eppendorf), spread on
nutrient agar plates (NA), and incubated at 30 °C
for 48-72 h. Bacillus-like colonies were subcultured
on new NA plates until pure cultures were obtained,
and they were kept at 4 °C for further identifi cation.
Th e reference strains, Bt kurstaki HD-1 and HD-73,
were kindly provided by Prof. Samir Jaoua (Centre
of Biotechnology of Sfax, CBS, Tunisia). Reference
strains and positive isolates were maintained in 20%
glycerol at −80 °C.
Light microscopy
Cells from Bacillus-like colonies were examined
for the presence of parasporal inclusion bodies by
light microscope. Bacteria from 1 of these colonies
were cultured in 5-mL aliquots of T3 medium
(Travers et al. 1987) at 30 °C in an orbital shaker at
200 rpm for 72 h. Resulting cultures were prepared
H. AMMOUNEH, M. HARBA, E. IDRIS, H. MAKEE
423
as fi lms on slides and heat fi xed, then stained with
Coomassie Brilliant Blue, as described by Rampersad
et al. (2002). Slides were observed with bright-fi eld
microscopy using a 100× oil immersion objective.
H serotyping
Th e serological identifi cation of the local Bt
isolates was carried out using only 2 reference H
antisera (H3a3b for Bt kurstaki and H5a5b for B.
thuringiensis galleriae). Reference H antisera were
obtained from Wuhan Institute of Virology, China.
Serotype was determined by tube agglutination tests
using diluted antisera, as described by Lecadet et
al. (1999). Tests were repeated twice to confi rm the
results.
Preparation of the spore-crystal mixture for the
bioassay
Isolates with parasporal bodies were grown in
suspension following the method of Carozzi et al.
(1991) with some modifi cations. In 500-mL fl asks,
100 mL of T3 medium was inoculated with 1 loop
of bacteria and shaken at 200 rpm for 5 days at 30
°C. At the end of this incubation, the majority of
the population was in the form of spores-crystals, as
monitored by light microscope. To remove exotoxins,
pellets (spores and parasporal crystal proteins) were
washed twice in 20 mL of sterile distilled water (SDW)
and centrifuged at 9700 g for 10 min. Pellets were
then processed through a freeze drier (temperature:
−50 °C, vacuum pressure: 0.1 mbar), and the resulting
dry powders were assayed for biological activity. Th e
supernatants of toxic samples were autoclaved (121
°C for 10 min) and subsequently used in toxicity
assays to check for the presence of thuringiensin
(β-exotoxin).
Insect rearing
All lepidopteran larvae used in the bioassays
were obtained from our laboratory colony insects.
E. kuehniella larvae were reared on wheat meal at
25 ± 1 °C without humidity control (Marec 1990). P.
operculella larvae were reared on waxed potato slices
at 25 ± 1 °C, 70 ± 5% RH, and a photoperiod of 12:12
h (L:D), as described by Saour and Makee (1997). C.
pomonella larvae were reared on artifi cial media at
25 ± 1 °C, 70 ± 5% RH, and a photoperiod of 16:8
h (L:D), as outlined in Makee (2005). Laboratory-
reared C. quinquefasciatus larvae were obtained
from the Wuhan Institute of Virology, China (Lab of
Biological Control of Arbovirus Vectors).
Toxicity tests and bioassays
A 2-step procedure was followed to evaluate the biological activity of Bt isolates against lepidopteran larvae. First, a screening to select those with toxicity against E. kuehniella, P. operculella, and C. pomonella larvae was carried out; then the toxicities were quantifi ed by determining the lethal concentration to kill 50% (LC
50) of the E. kuehniella larvae. For
screening tests, third-instar larvae of E. kuehniella and fi rst-instar larvae of P. operculella and C. pomonella were treated with single doses of spore-crystal mixture (SCM). Bt kurstaki HD-1 and HD-73 were used as standards since their toxicity against these insect species is well known (Federici et al. 2006), and SDW was included as a negative control. Concentrations of all mixtures were adjusted to 1 mg mL-1, the level at which the standard strains produced an insect mortality of 100%. First, 20 larvae were transferred to a 5-cm petri dish containing 1 g of larval food and mixed with 500 μL of SCM. Aft er 5 days, the average number of dead larvae was recorded, and isolates were characterized as very toxic (100% mortality) or nontoxic (0% mortality). Toxic isolates were subjected to quantifi cation of their activities by determining the LC
50 against E.
kuehniella. Bioassays were performed basically as described above, including 10-fold serial dilutions that ranged from 200 to 0.2 μg SCM g-1 wheat meal. For each dose, 5 replicates were prepared, with 10 larvae in each replicate. A total of 500 larvae for each isolate were used, and mortality was recorded aft er 5 days. Bioassay results were processed and analyzed by means of the SOFTTOX computer program (WindowChemTM), which is based on the probit analysis method (Finney 1971). To evaluate the biological activity of the isolates against a dipteran species, SCM was tested against C. quinquefasciatus larvae using disposable plastic cups, and the test medium was prepared by adding 1 mL of bacterial suspensions in 100 mL of sterile water. Separately, 20 larvae of third-instar C. quinquefasciatus were introduced into each cup. Following 24 and 48 h of exposure at room temperature with no food, larval
Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against
some insect pests
424
mortality was recorded. Larvae exposed to water only and bacterial suspensions of Bt cry- (a strain with no cry genes was kindly provided by Prof. Samir Jaoua, CBS, Tunisia) served as the negative control. Larvae exposed to water with Bt israelensis served as a positive control.
Identifi cation of cry genes
Screening for cry genes was carried out using PCR. Family-specifi c primer pairs directed toward the identifi cation of some of the main groups of cry genes were used: cry1 (Lep1A, Lep1B; Lep2A, Lep2B; and Un1F, Un1R), cry2 (Un2F, Un2R), cry3 (Col1A, Col1B and Un3F, Un3R), cry4 (Dip1A, Dip1B and Un4F, Un4R), cry7 and cry8 (Un7-8F, Un7-8R), cry9 (Un9F and Un9R), cry10 (10A5, 10A3), cry11 (EE11a(d), EE11a(r) and 11A5, 11A5), cry13A (spe-cry13d, spe-cry13r), cry15A (34C, 34D), cry16A (DA5c, CR3c), cry17A (CR8, OX7as), and cry12, cry14, cyr18, cry19, cry20, cry21, cry22, cry23, cry25, cry26, cry27, and cry28 (F, R) (Carozzi et al. 1991; Ben-Dov et al. 1997, 1999; Porcar and Juárez-Pérez 2003). To obtain bacterial DNA, a loopful of cells from an LB plate grown overnight was suspended in 100 μL of SDW in a microfuge tube and vortexed. Th e mixture was frozen at −20 °C for 20 min and then boiled for 10 min to lyse the cells. Th e mixture was then centrifuged briefl y at 9700 g, and 5 μL of the upper phase was added as a template to 25-μL PCR reactions. PCR mixtures were prepared using 0.4 μM of each primer; 1 U of Taq DNA polymerase (Promega); 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Promega); and 2 mM MgSO
4 and 3%
dimethyl sulfoxide (DMSO). Amplifi cation was done in a Bio-Rad T gradient thermocycler under the following conditions: a 5 min denaturation step at 95 °C followed by 30 amplifi cation cycles (1 min at 95 °C, 1 min at 48-58 °C, and 1 min at 72 °C) and an extra extension step of 10 min at 72 °C. PCR products were separated on a 1%-2% agarose gel, to which ethidium bromide was added, and photographed under UV light.
Rapid analysis of crystal proteins
A single colony was removed from an LB agar plate, inoculated into nutrient broth, and incubated at 30 °C for 72 h with continuous shaking at 200 rpm. Next, 2 mL was centrifuged at 9800 g for 10 min and resuspended in 1 mL of ice-cold 0.5 M NaCl. Cells were centrifuged at 9800 g for 5 min; the resulting pellet was resuspended in 1% SDS
and 0.01% mercaptoethanol, boiled for 10 min, and recentrifuged at 9800 g for 10 min. Th e supernatant was removed and analyzed by 12% SDS-PAGE, which was conducted at 36 mA for 1 h in a mini-Protean II apparatus (Bio-Rad), as described by Laemmli (1970). Gels were stained in 0.1% Coomassie Brilliant Blue R-250. Th e protein masses were determined by comparison with molecular weight standards (Fermentas SM0431).
Results
In total, 200 soil samples were collected from all Syrian provinces. Sampling results are shown in Figure 1 and the Table. Following observations under a light microscope, a total of 525 Bacillus-like colonies and 25 isolates (4.7%) were putatively identifi ed as Bt since the distinctively staining proteinaceous crystals characteristic of the species were observed. Since Bt index is defi ned as the number of identifi ed Bt colonies divided by the total number of Bacillus-like colonies examined, we obtained a Bt index of 0.047 (Table). Geographically, the west-central and northeastern Syrian regions showed the highest percentage of soil samples with Bt isolates (20.58% and 17.6%, respectively), followed by the northern, southern, and western regions (6.66%, 2.85%, and 0%, respectively) (Figure 1 and Table).
Figure 1. Map of Syria showing the locations of the sites from
which the soil samples were collected (dots). Stars
indicate sites from which toxic Bacillus thuringiensis
was isolated.
H. AMMOUNEH, M. HARBA, E. IDRIS, H. MAKEE
425
Selective bioassay with a single-dose showed that all obtained Bt isolates were very toxic (100% mortality) to E. kuehniella, P. operculella, and C. pomonella larvae. Despite the high concentrations of local isolates tested, no mortality was observed in any of the C. quinquefasciatus larvae, apart from the Bt israelensis (positive control) within 24 h. However, aft er 48 h only the larvae in the negative controls were alive at a rate of 100%; mortality was more than 50% in all other treatments. Moreover, in all of the 25 isolates, nonspecifi c thuringiensin (β -exotoxin) activity was absent, as their autoclaved supernatants were not toxic to E. kuehniella larvae.
All 25 toxic isolates were selected for further characterization, including crystal morphology, more extensive bioassays, and analysis of cry gene content and Cry protein patterns. Very clear bipyramidal- and cuboidal-shaped crystals were observed in all isolates aft er staining and examining the cells by light microscopy (Figure 2). Th e mean estimated
LC50
values of the tested isolates against E. kuehniella larvae varied from 8.4 to 97.6 μg g-1 (Figure 3). Out of the 25 isolates, 12 with LC
50 values ranging from 8.4
to 46.8 μg g-1 showed a toxicity against E. kuehniella larvae that was equal to or higher than that of the Bt. kurstaki HD-73 strain (LC
50 46 μg g-1). However, only
5 isolates (SSy125-c, SSy60-b, SSy141-c, SSy126-c, and SSy111-c) were more toxic than the Bt kurstaki HD-1 strain (LC
50 20.8 μg g-1) (Figure 3).
Profi les of all PCR products were compared with those of standard strains (e.g. Bt kurstaki HD-1). An isolate was considered to contain a determined gene only when the amplifi cation product was of the expected size. Analysis of the local Bt isolates indicated that all of the tested isolates contained several cry1 genes as well as cry2. All tested isolates produced approximately 277 bp using the general cry1 primers (Uni1F, Uni1R). Additionally, when the Lep1A, Lep1B and Lep2A, Lep2B primers were used, all of the tested isolates produced the expected
Table. Isolation of lepidopteran-toxic Bacillus thuringiensis from Syrian soil samples.
RegionProvinces
No. of soil
samples
analyzed
No. of
samples with
Bt (%)
No. of
Bacillus-like
colonies examined
No. of Bt
isolates
obtained
Bt index†
Southern
Qunaytirah 5 0 14 0 0
Suwayda 15 0 27 0 0
Damascus 15 1 (6.66) 31 1 0.032
West-central
Homous 12 1(8.33) 18 1 0.055
Palmyra 10 3 (30) 27 3 0.111
Hamah 12 3 (25) 27 3 0.111
WesternTartous 13 0 35 0 0
Latakia 8 0 24 0 0
NorthernAdleeb 9 0 26 0 0
Aleppo 21 2(9.5) 51 2 0.039
Northeastern
Raqqa 32 4 (12.5) 85 4 0.047
Dier Ezzor 33 6 (18.18) 104 6 0.057
Hassaka 20 5 (25) 56 5 0.089
Total 200 25 (12.5) 525 25 0.047
†Bt index: No. of identifi ed B. thuringiensis colonies divided by the total number of Bacillus-like colonies examined.
Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against
some insect pests
426
a b
c d
Figure 2. Light microscope photomicrographs of sporulated cultures of 3 local isolates
of Bacillus thuringeinsis: a) SSy60-b, b) SSy125-c, c) SSy126-c, and d) reference
strain B. thuringiensis kurstaki HD-1, showing typical parasporal inclusion.
Large arrow: bipyramidal, small arrow: cuboidal, arrow and dot: spores, star:
sporulated cell, bar = 5 μm.
Figure 3. LC50
values (μg g-1) of 25 local isolates of Bacillus thuringeinsis against Ephestia kuehniella larvae. HD-1 and HD-73: B.
thuringiensis kurstaki HD-1 and HD-73 (controls), SSy: local isolates collected from Syrian soil samples; bars represent the
95% upper and lower fi ducial limits.
0
10
20
30
40
50
60
70
80
90
100
110
HD
-1
HD
-73
SSy5
2-b
SSy5
3-b
SSy5
9-b
SSy6
0-b
SSy6
1-b
SSy6
2-b
SSy 63-
b
SSy6
9-c
SSy 77
SS y108-c
SS y111-c
SS y112
-c
SS y124
-c
SS y125
-c
SS y126
-c
SS y127
-c
SS y135
-c
SS y141
-c
SSy1
78-c
SSy1
87-c
SSy1
88-c
SS y189-b
SS y195-c
SS y199-c
SS y200-c
Isolates
LC
50
H. AMMOUNEH, M. HARBA, E. IDRIS, H. MAKEE
427
sizes of PCR products of cry1Aa/cry1Ac (490/986
bp). Only 13 isolates produced PCR products of
cry1Ab (490/908 bp) of the expected sizes (Figure
4a). Moreover, all of the tested isolates produced PCR
products of the expected size (approximately 700
bp) when the general cry2 primers (Uni2F, Uni2R)
were used (Figure 4b). On the other hand, none of
the tested isolates produced the expected sizes of any
PCR products of cry3, cry4, cry7, cry8, cry9, cry10,
cry11, cry12, cry13A, cry14, cry15A, cry16A, cry17A,
cyr18, cry19, cry20, cry21, cry22, cry23, cry25, cry26,
cry27, or cry28 genes when appropriate primers for
detection of these genes were used. All local Bt isolates
were further characterized by SDS-PAGE. Results
indicated that all isolates synthesized 2 protein types
with molecular weights of approximately 130 and 65
kDa (Figure 5).
Flagellar serotyping revealed that 4 isolates
(SSy125-c, SSy141-c, SSy111-c, and SSy112-c)
belonged to serotype kurstaki, 15 isolates gave
negative reactions with the 2 tested antisera, and 6
isolates (SSy52-b, SSy53-b, SSy62-b, SSy63-b, SSy77,
and SSy108-c) were not typable isolates.
Discussion
Screening the environment for new and highly
potent strains of Bt has become inevitable as one of
the strategies for insect resistance management. Many
reports on the frequent occurrence of Bt isolates in
the natural environment showed the high possibility
of isolating a novel strain. Th e establishment of
worldwide collection has contributed to our current
knowledge of the ecological distribution of this
bacterium in nature. Most of these studies proved
the ubiquity of Bt, which is found in any type of soil
(Dulmage and Aisawa 1982; Martin and Travers 1989;
Bernhard et al. 1997; Hossain et al. 1997; Bravo et al.
1998). In this study, various types of soil samples that
had not been previously treated with Bt formulations
were collected from diff erent Syrian regions. Th e
discovery of Bt isolates that displayed insect toxicity
indicates the existence of local isolates. Our results
clearly revealed that Bt was ubiquitously distributed
in Syrian soils (Figure 1). Furthermore, 12.5% of
Syrian soil samples analyzed were found to contain Bt
isolates (Table). Th is value is considerably low when
compared with other studies that found percentages
Figure 4. Detection of Bacillus thuringiensis isolates’ insecticidal genes with a) cry1 general primers (Lep1A, Lep1B and Lep2A, Lep2B)
and b) cry2 general primers (Uni2F, Uni2R). Lane M: DNA 1 Kb ladder, lane 1: SSy52-b, lane 2: SSy53-b, lane 3: SSy59-b,
lane 4: SSy60-b, lane 5: SS61-b, lane 6: SSy62-b, lane 7: SSy63-b, lane 8: SSy77, lane 9: SSy108-c, lane 10: SSy111-c, lane 11:
Ssy112-c, lane 12: SSy125-c, lane 13: SSy127-c, lane 14: SSy135-c, lane 15: SSy69-c, lane 16: SSy124-c, lane 17: SSy126-c, lane
18: SSy141-c, lane 19: SSy178-c, lane 20: SSy187-c, lane 21: SSy188-c, lane 22: SSy189-b, lane 23: SSy 195-c, lane 24: SSy199-c,
and lane 25: SSy200-c. +: Bt kurstaki HD-1 (positive control), -: Bt israelensis (negative control).
a b
Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against
some insect pests
428
between 20% and 70% (Martin and Travers 1989; Chilcot and Wigley 1993; Ohba and Aratake 1994; Vásquez et al. 1995). Th ese results could be attributed to the fact that a comparatively small area was screened. Martin and Travers (1989), for example, estimated a value of 71.6% from soil samples collected worldwide. Th is is based on the hypothesis that larger areas embrace more kinds of habitat, thus supporting more species (Strong et al. 1984). However, Ohba et al. (2002) reported that the frequency of Bt-positive soil samples averaged between 9.5% and 16.9% in the oceanic islands of Japan. On the other hand, the high abundance of Bt isolates in the west-central and northeastern regions of Syria may be due to the fact that most of the soil samples collected from these regions were uncultivated soils. In contrast, most of the soil samples collected from other regions were cultivated soils, and the low abundance of Bt in these samples may be due to the presence of disinfectants and waste chemicals. Th is is in agreement with the fi ndings of Martin and Travers (1989), who also showed that Bt was more productive in some areas than others.
Th e screening for local Bt isolates was carried out by the acetate enrichment method described by Travers et al. (1987). Th ese authors claimed that germination of Bt spores is selectively inhibited in the presence of sodium acetate, whereas most other spore formers are germinated. Selection of Bt was
attempted by eliminating germinated cells through
15 min of heat treatment at 80 °C. We observed only
marginal selection by acetate over other bacilli (Bt
index = 0.047), and that is consistent with a previous
study reported by Bernhard et al. (1997).
Bt is not normally toxic to insect larvae that live
in the soil (Martin and Travers 1989), but it is toxic
to insects that are aerial and waterborne larvae (Wu
and Chang 1985). Consequently, E. kuehniella, P.
operculella, C. pomonella, and C. quinquefasciatus
larvae were chosen for the primary toxicity tests. A
series of bioassays were performed by providing the
tested larvae with food containing the spores and
crystals. Spores and crystals were both included
in the suspensions because they produce a higher
level of mortality than either crystals or spores
alone (Crickmore 2006). Quantitative bioassay
results showed that all local Bt isolates were toxic
to the tested lepidopteran larvae. Th is result was
consistent with previous reports (DeLucca et al.
1981; Ohba and Aizawa 1986a, 1986b; Martin
and Travers 1989). Chilcott and Wigley (1993)
showed that the percentage of isolates obtained
from soil with toxicity against lepidopteran larvae
ranged from 37% to 88%. Similarly, Iriarte et al.
(1998) reported that most of the Bt isolates showed
insecticidal activity (above 25% mortality) against
some lepidopteran species.
Figure 5. SDS-PAGE of spore-crystal mixture from some local B. thuringiensis isolates. Lane M: molecular marker given in kDa
(Fermentas), lane 1: SSy52-b, lane 2: SSy53-b, lane 3: SSy59-b, lane 4: SSy60-b, lane 5: SSy61-b, lane 6: SSy62-b, lane 7: SSy63-b,
lane 8: SSy77-c, lane 9: SSy108-c, lane 10: SSy111-c, lane 11: Ssy112-c, lane 12: SSy125-c, lane 13: SSy127-c, lane 14: SSy135-c,
lane 15: SSy141-c, and lane 16: SSy178-c. +: Bt kurstaki HD-1 (positive control).
H. AMMOUNEH, M. HARBA, E. IDRIS, H. MAKEE
429
E. kuehniella is one of the major pests in industrial
fl our mills in temperate climates (Jacob and Cox
1977) and causes serious damage in amylaceous
products. Apart from direct infestation, larvae feces
and webbings spoil the product. For the control of
stored-cereal species, including E. kuehniella, the
main categories of pesticides used are fumigants
and residual grain protectants. However, the use
of these substances is being reduced for health and
environmental safety reasons (Arthur 1996; Bell
2000). Consumer demand for residue-free food
necessitates the evaluation of alternative, reduced-
risk control methods. Insect pathogens are among the
most promising alternatives to traditional pesticides
in stored-product protection. Bt has been approved
as a grain protectant in the United States and is
commercially available for the control of Indian
meal moth larvae. Eff ective control using Bt has been
reported against lepidopteran larvae attacking stored
products (Brower et al. 1995; Moore et al. 2000).
Interestingly, our qualitative bioassay data showed
that most of the tested local Bt isolates were highly
toxic to E. kuehniella larvae. Remarkably, SSy125-c
and SSy60-b were 2 and 5 times more toxic to E.
kuehniella larvae than Bt kurstaki HD-1 and HD-73
strains, respectively (Figure 3). All local lepidopteran
toxic isolates had very low toxicity against dipteran
larvae and produced both bipyramidal and cuboidal
crystal proteins. Th e results are in agreement with
those of Ohba and Aizawa (1986a, 1986b), who
suggested a possible relationship between the shape
of the parasporal bodies and toxicity. Most reports
showed that strains producing bipyramidal crystal
proteins exhibited toxicity only to Lepidoptera and
were associated with Cry1 toxins, whereas cuboidal
crystal proteins exhibited toxicity to Lepidoptera
and Diptera and were most probably associated with
Cry2 toxin (Federici et al. 2006).
One of the most important factors aff ecting diff erences in toxicity is the cry gene content. In general, the type of cry genes present in a strain correlates to some extent with its insecticidal activity. Th us, the identifi cation of the gene content in a strain can be used to predict or confi rm its insecticidal potential. Th e presence of cry1 and cry2 was confi rmed by PCR in all tested isolates using primers
designed by several authors (Carozzi et al. 1991; Ben-
Dov et al. 1997). Th e Bt kurstaki HD-1 strain was used
as a reference strain because it harbors the cry1Aa,
cry1Ac, cry1Ab, cry1Ia, and cry2 genes. Th e protein
profi le of the crystal protein extract from each of the
local isolates was compared to that of the Bt kurstaki
HD-1 strain. All profi les were similar and showed 2
bands of around 130 and 65 kDa, which supported
the idea that cry1 and cry2 genes were expressed in
all tested isolates (Figure 5).
Interestingly, 4 local isolates (SSy125-c, SSy141-c,
SSy111-c, and SSy112-c) identifi ed as Bt kurstaki
showed higher toxicity than the standard strains
(Bt kurstaki HD-73 or HD-1), which are the active
ingredients of commercial preparations commonly
used as bioinsecticides (Federici et al. 2006).
Th erefore, the results of this study confi rm the
value of the continuing isolation of new Bt strains,
including those belonging to the previously described
serotypes, from new areas. Th e cultures of 6 local
isolates were agglutinated spontaneously in the
absence of the specifi c antiserum when subjected to
the conditions required for the agglutination tests. As
a result, serotyping of these isolates was impossible,
and they are not considered typable isolates.
Lecadet et al. (1999) reported that untypable strains
make up almost 3% of the Bt in the International
Entomopathogenic Bacillus Centre’s collection. Th e
reasons for the spontaneous agglutination of these
strains are not clear, but this the only situation in
which the H-classifi cation is completely useless.
Th erefore, the H-classifi cation should be used
together with other methods, including biochemical
and molecular methods, for classifying Bt strains
(Lecadet et al. 1999).
Bt has been used commercially in the biological
control of insect pests for the last 4 decades. Our
results indicate the presence of Bt isolates in Syrian
soil that show insecticidal activity. Bioassay results
confi rmed that the lepidopteran-toxic local Bt
isolates vary in their pathogenic activity against the
same insect. Th e presence and the expression of
cry1 and cry2 genes were confi rmed in all isolates.
Th erefore, the diff erences in toxicity among these
isolates could be a result of the expression level or
the diff erence in the copy number of the cry1 or cry2
genes. Future studies will deal mainly with the most
Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against
some insect pests
430
toxic local isolates. Th ese studies will focus on the
molecular characterization of cry1 and cry2 genes
by PCR-sequencing and real time PCR, testing the
bioactivity of isolates against diff erent pest insects
of other species, and investigating their use in the
production of novel biopesticides or the engineering
of pest-resistant plants. Th is could play a crucial
rule in controlling important insect pests and lead
to a reduction in the intensive use of some chemical
insecticides in Syria.
Acknowledgments
Th e authors would like to thank the Director General of the Atomic Energy Commission of Syria and the head of the Molecular Biology and Biotechnology department for their continuous support throughout this work. A scientifi c visit for H. Ammouneh to the Lab of Biological Control of Arbovirus Vectors, Wuhan Institute of Virology, China was supported by the TWAS-CAS fellowship program.
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