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Use of microbial agents is an important tool for insect control because of their virtual specificity and lower environmental impacts. A total of four soil samples were collected from different localities across the black sea region in Turkey to isolate native Bacillus thuringiensis (Bt) strains. Sodium acetate-(0.25 M)-selection heat-pasteurization, and 50% ethanol treatment methods were used for Bt isolation. Characterization of Bt isolates based on their morphological, physiological and biochemical parameters. PCR analysis was performed, using novel general and specific primers for cry genes (cry4A and cry10 genes) that encoding proteins active against mosquitoes. Based on results, the isolate Bt22.19 showed the highest larvicidal effect against 3 rd instar C. pipiens larvae. Bt 22.19 was isolated from soil samples in hazelnut orchards. The isolated strains had cry4A and cry10 genes. Bt isolates displayed highest similarities with the Bacillus thuringiensis subsp. israelensis regarding to the presence of cry genes. Obtained results are a promising introduction for further studies on evaluation of the potential usefulness of isolate Bt22.19 crystals for mosquito's control.
Egyptian Journal of Biological Pest Control, 25(2), 2015, 439-444
Isolation and Characterization of Bacillus thuringiensis Isolated from Soil
and their Possible Impact on Culex pipiens Larvae
Aksoy*, H. M.; I. Saruhan*; I. Akca*; Y. Kaya**; H. Onder***; M. Ozturk* and O. Aker*
*Ondokuz Mayis University, Agricultural Faculty, Plant Protection Department, Samsun, 55139, Turkey.
**Ondokuz Mayis University, Agricultural Faculty, Plant Biotechnology Department, Samsun, 55139, Turkey.
***Ondokuz Mayis University, Agricultural Faculty, Animal Science Department, Samsun, 55139, Turkey.
(Received: August 16, 2015 and Accepted: September 22, 2015)
Use of microbial agents is an important tool for insect control because of their virtual specificity and lower environmental
impacts. A total of four soil samples were collected from different localities across the black sea region in Turkey to
isolate native Bacillus thuringiensis (Bt) strains. Sodium acetate-(0.25 M)-selection heat-pasteurization, and 50% ethanol
treatment methods were used for Bt isolation. Characterization of Bt isolates based on their morphological, physiological
and biochemical parameters. PCR analysis was performed, using novel general and specific primers for cry genes (cry4A
and cry10 genes) that encoding proteins active against mosquitoes. Based on results, the isolate Bt22.19 showed the
highest larvicidal effect against 3rd instar C. pipiens larvae. Bt 22.19 was isolated from soil samples in hazelnut orchards.
The isolated strains had cry4A and cry10 genes. Bt isolates displayed highest similarities with the Bacillus thuringiensis
subsp. israelensis regarding to the presence of cry genes. Obtained results are a promising introduction for further studies
on evaluation of the potential usefulness of isolate Bt22.19 crystals for mosquito’s control.
Key words: Bacillus thuringiensis, Culex pipiens, larvicidal effects.
The desire for mosquito control increased
significantly. Mosquitoes have the potential and
lethal capacity to kill more than 1 million victims a
year around the world. Culex spp. is a common
mosquito species throughout the world including
Turkey (Harbach, 2012). Culex spp. transmits
pathogens causing human diseases throughout the
world that include dengue fever, malaria and yellow
fever (Almeida et al., 2008). Common insecticides
have been applied for mosquitos’ elimination has
given rise to problems for human and environment.
The most useful methods for controlling these
diseases based on vector control that is mainly
accomplished by using synthetic insecticides. Use of
entomopathogenic bacteria as biolarvicides is a favor
alternative for insect controls (Regis et al., 2001). It
has been safely used against species of the orders
Lepidoptera, Coleoptera, and Diptera for the last 50
years (Roh et al., 2007). Bacillus thuringiensis (Bt) is
a rod-shaped, aerobic, gram positive and spore-
forming bacterium. It forms a parasporal crystal
during sporulation (Höfte and Whiteley, 1989). These
parasporal crystals consist of insecticidal-endotoxins
with specific toxicity towards a variety of
lepidopteran, dipteran and coleopteran larvae (Gill et
al., 1992) and called Cry proteins, expressed by the
cry genes (Schnef et al., 1998). Number of known Bt
strains active on Diptera is growing (Guerchicoff et
al., 1997). B. thuringiensis subsp. israelensis cry and
cyt genes encode dipteran-active toxins: Cry4A,
Cry4B, Cry10A, and Cry11A, Cyt1A, and Cyt2B
(Guerchicoff et al., 1997 and Salehi et al., 2008). To
date, Bt strains have been isolated from many
bacterial habitats, including plant tissue, soil, insects,
and water (Ichimatsu et al., 2000 and Iriarte et al.,
2000), free-living animals (Swiecicka et al., 2002).
The aim of this study was, to find Bt isolates from
different soils in Samsun Province, Turkey, to
characterize these strains by molecular methods and
to determine their larvicidal activity against C.
pipiens larvae.
Soil samples
Soil samples were collected in glass tube from
surface to a depth of 10 cm in the hazelnut orchards.
A total of 37 soil samples were collected from the
black sea region of Turkey from April, 2013 to June,
2013. This origin of samples had not been previously
treated with any Bt biopesticides. The collected
samples were kept at about 4°C in an incubator till
been used for bacterial isolation.
Isolation of bacteria
The soil samples of 1g were suspended each in
10ml 0.85% NaCl and heated with shaking at 70°C
for 10min. Aliquots of 100μl of suspension were
plated onto nutrient agar (Difco nutrient broth
solidified with agar, 10 g l-1). The plates were then
incubated at 30±2°C for 48h, then the culture stained
with amino black and Ziehl’s carbol fuchsin and
examined under a standard light
microscope. Bt isolates were selected when black
crystals dyed black were noticed (Yu et al., 1991).
Biochemical characteristics
Morphological, physiological and biochemical
Table (1): Characteristics of general and specific primers for cry1A, cry2, cry3A, cry4A, cry6A, cry8, cry9,
cry10, cry11, cry19, cry21, cry22, cry30, cry40, cry54, cry62A, and cyt1A genes
Primer Sequence* Positions Gene(s) Product size
recognized (bp)
Cry1A F 5’-GAGGGAATGGCACGGGTTTA-3’ 893-912 cry1A 689
Cry2 F 5’-GTAGTGGACCACAGCAGACC-3’ 836-855 cry2 336
Cry3A F 5’-GTGGAGCGCTTGTTTCGTTT-3’ 54-273 cry3A 528
Cry4A F 5’- ACGGGGATTTTGAATCGGCT -3’ 2.276-2.295 cry4A 940
Cry6A F 5’-GGGGAAAGTAGTCCAGCTCA-3’ 888-907 cry6A 465
Cry8 F 5’-GCGTTAATCCAGCTGCGATT-3’ 101-120 cry8 1294
Cry9 F 5’-TCTATGGGGCAAGATGGGGA-3’ 656-675 cry9 1656
Cry10 F 5’-ATAAATGGGAGCCAGCACGT-3’ 470-489 cry10 1142
Cry11 F 5’-ATAGGGAAATGGGCGGCAAA-3’ 145-164 cry11 1406
Cry19 F 5’-CCACAAATGCCCATGCGAAA-3’ 103-122 cry19 1422
Cry21 F 5’-ACACCCTGCTGAACGATCTG-3’ 83-102 cry21 287
Cry22 F 5-CAAGCAGGAGCAATTGCAGG-3’ 151-170 cry22 364
Cry30 F 5’-TCCAGGAGCAGCTGTAGGAT-3’ 252-271 cry30 1394
Cry40 F 5’-TGTGGGAATCAACCTCGAGC-3’ 149-168 cry40 946
Cry54 F 5’-CCGGAGTTAGTGCAGGTGTT-3’ 197-216 cry54 1488
Cry62A F 5’-GGACCCTGCCACGATTAACA-3’ 687-706 cry62A 1301
Cyt1A F 5’-CCCTCAATCAACAGCAAGGG-3’ 57-76 cyt1A 593
* Position at 5’ end of direct and reverse primers for each PCR primer pair. F and R forward and reverse primers, respectively.
characteristics of the Bt isolates were determined
according to the standard methods recommended in
Bergey’s Manual of Systematic Bacteriology (Sneath
et al., 1984).
Determination of specific cry genes
To identify the larvicidal genotypes of Bt isolates,
seventeen pairs of primers were designed to detect the
presence of the cry and cyt genes through PCR of their
conserved regions. All primers were designed using
Geneious 7 software (Biomatters, Auckland, New
Zealand) based on manual identification of specific
cry and cyt genes regions of the sequence alignment.
BLAST analysis was also performed among chosen
cry and cyt genes sequences in NCBI database to
confirm the specificities of the primer sets. The full
list of primers is provided in table (1). The PCR
mixture contained 1 μl of DNA in a total volume of
10 μl containing 200 nM concentrations of each
primer constituted in 1x BioMix Red (Bioline,
Boston,F Massachusetts, United States) PCR reaction
buffer. The PCR mixture contained 1 μl of DNA in a
total volume of 20 μl containing 200 nM of each
primer in 1× reaction buffer. PCR amplifications
were performed in a T100 thermal cycler (Bio-
Rad) with the following touchdown cycling
conditions: 4 min at 94C, 24 cycles of 0.30 min at
94C, 0.30 min at 65C with a 1C decrease per cycle,
and 1.30 min at 72C, followed by 10 cycles of 0.30
min at 94C, 0.30 min at 56C, and 1.30 min at 72C,
ending with a 16C hold. PCR products were
monitored on a 1.0% agarose gel in 1x TAE buffer at
100V for about 60 min and checked their quality, size
and yield.
Bioassays for mosquito larvae
The treatments consisted of:
(i) B. thuringiensis isolates 22.19;
(ii) B. thuringiensis isolates hma 5;
(iii) B. thuringiensis isolate hma7;
(iv) B. thuringiensis isolates bmeg;
(v) Positive control - commercial biopesticide of B.
thuringiensis (VectoBac) WG; and
(vii) Negative control.
Bt isolates were grown onto nutrient agar (Difco)
at 30±2°C for 24 h. The cells were then harvested and
suspended in sterile distilled water at concentrations
of [101, 103, 106 and 109 cfu/ml, colony-forming
units/ml] and heat shocked (10 min, 70C). Different
doses of 8,000; 16,000; and 32,000 international toxic
units (ITU)/mg of commercial biopesticide VectoBac
WG was used as positive control. Untreated box was
negative control. Third-instar larvae were used for all
bioassays in 50ml of tap water in plastic cups
according to the standard bioassay procedure (WHO,
2005). Each bioassay was independently performed
three times in duplicate. C. pipiens larvae were
provided by Ondokuz Mayis University, Agricultural
Faculty, Plant Protection Department, Entomology
Lab. (Samsun, Turkey). Mortality was recorded after
6, 12, 24 and 48h.
Data analysis
Kolmogorov-Smirnov one sample test used to
examine normal distribution and Levene test for equal
variance (homosceasticity) assumption (Onder,
Biochemical characteristics
Four of gram-positive and spore forming bacilli
were isolated from soil. Morphological, physiological
and biochemical characteristics of isolates are shown
in table (2). All isolates showed a positive reaction in
aerobic growth and gelatin liquefaction tests.
Biochemical data revealed that a negative reaction in
arabinose and mannitol. The isolates of Bt22.19 and
Btbmeg could grow in sodium chloride solutions at
concentrations ranged 5-10%, while the isolates of
Bthma5 and Bthma7 that couldn't grow in NaCl
solution at concentration of 10%.
Determination of specific cry genes
The mosquitocidal genotypes of Bt were
determined through PCR designed primers by using
Geneious 7 software (Biomatters, Auckland, New
Zealand) for the cry1A, cry2, cry3A, cry4A, cry6A,
cry8, cry9, cry10, cry11, cry19, cry21, cry22, cry30,
cry40, cry54, cry62A, and cyt1A genes. Four Bt
isolates obtained from soil samples carried cry4A, and
cry10 genes. The isolates exhibited the 940 bp and
1142 bp fragments of the cry4A and cry10 genes,
Table (2): Morphological, physiological and
biochemical characteristics of Bt isolates
Bacillus thuringiensis isolates
Gram staining
Aerobic growth
Gelatin liquefaction
Acid from Arabinose
Growth at 5%
Growth at 30C
*(-) negative reaction, (+) positive reaction, (G+) Gram positive.
respectively, that encode the crystal protein toxic to
mosquitoes. None of the isolates had cry5, cry6A,
cry8, cry9, cry19, cry21, cry22, cry30, cry40, cry54,
cry 62A and cyt1 genes. BLAST analysis indicated
that it corresponded to the cry4A and cry10 genes
(100% identity). The Bt serovar strain BRC-LLP29
was used as a control for cry4A, and cry10 genes (data
not shown).
Bioassay results indicated that the Bt isolates were
toxic against the 3rd instar larvae of C. pipiens.
Percentage mortality of Bt isolates are shown in table
(3). At bacterial concentration of 106 cfu/ml within
48h, percentages of mortality ranged 26.67 - 98.33%,
where they were 98.33, 36.67, 31.67 and 26.67% for
the isolates Bt22.19, Btbmeg, Bthma7 and Bthma5,
respectively (Figure 1). Insignificant differences were
recorded among mortality rates in the isolates of
Btbmeg, Bthma7 and Bthma5. At concentration of
109 cfu/ml, the mortality percentage ranged between
33.33 - 100.0%, where the highest mortality (100.0%)
was recorded at the isolate Bt22.19. Btbmeg, Bthma7
and Bthma5 isolates caused the least larval death,
where the percentage mortality ranged of 33.33 -
40.0% (Figure 2). Highest mortality % were 100,
88.33, 78.33, and 50.0%, obtained at the treatment of
Bt22.19 isolates at the concentrations of 109 cfu/ml,
while treatment with the commercial biopesticide
Wertobag gave highest mortality by 100, 100, 93.33
and 66.67% at the concentration of 32000, ITU/mg
within 48, 24, 12 and 6h, respectively (Figure 3).
Synthetic insecticides have been associated with
human health problems such as cancer, liver damage
and birth defects beside environmental problems.
Some of the microorganisms like, Bacillus spp. in
controlling insects that transmit human diseases, is
well established. Accordingly, the present work was
proposed to isolate and characterize Bt isolates from
black sea region habitat and to test their effect against
Table (3): Effects of Bacillus thuringiensis isolates on mean percentage mortality of the 3rd instar larvae
0.00 ± 0.00g*
28,33 ± 1,67e
63,33 ± 4,41d
78,33 ± 1,67c
0.00 ± 0.00g
46,67 ± 1,67d
83,33 ± 6,01bc
88,33 ± 6,01bc
41,67 ± 4,41c
65.00 ± 5,77c
86,67 ± 1,67b
98,33 ± 1,67ab
50.00 ± 5,77b
78,33 ± 1,67b
88,33 ± 1,67b
100.00 ± 0.00a
0.00 ± 0.00g
5.00 ± 0.00ij
5.00 ± 0.00gh
10.00 ± 2,89hi
0.00 ± 0.00g
8,33 ± 3,33hij
10.00 ± 2,89fgh
16,67 ± 4,41gh
8,33 ± 1,67def
15.00 ± 0.00fg
21,67 ± 4,41ef
26,67 ± 4,41efg
13,33 ± 1,67d
20.00 ± 5,77ef
28,33 ± 6,01e
33,33 ± 1,67def
0.00 ± 0.00g
6,67 ± 1,67hij
11,67 ± 1,67fg
16,67 ± 1,67gh
0.00 ± 0.00g
13,33 ± 3,33fgh
16,67 ± 6,67efg
20.00 ± 7,64gh
5.00 ± 2,89fg
13,33 ± 1,67fgh
25.00 ± 2,89e
31,67 ± 4,41def
6,67 ± 1,67efg
26,67 ± 1,67e
28,33 ± 1,67e
40.00 ± 2,89d
0.00 ± 0.00g
6,67 ± 1,67hij
10.00 ± 2,89fgh
10.00 ± 2,89hi
0.00 ± 0.00g
10.00 ± 2,89hij
16,67 ± 4,41efg
23,33 ± 6,01fg
5.00 ± 0.00fg
15.00 ± 2,89fg
25.00 ± 2,89e
36,67 ± 3,33de
11,67 ± 1,67de
25.00 ± 2,89e
28,33 ± 6,01e
38,33 ± 4,41d
toxic unit-ITU)
38,33 ± 1,67c
63,33 ± 1,67c
75.00 ± 2,89c
96,67 ± 1,67ab
53,33 ± 1,67b
81,67 ± 1,67b
91,67 ± 1,67ab
100.00 ± 0.00a
66,67 ± 1,67a
93,33 ± 1,67a
100.00 ± 0.00a
100.00 ± 0.00a
0.00 ± 0.00g
3,33 ± 1,67j
0.00 ± 0.00h
0.00 ± 0.00i
(*) Mean followed by the same letters in each column are not significant.
Fig. (1): Mortality of Culex pipiens 3rd instar larvae
treated with Bacillus thuringiensis isolates for
different times at the concentration of 106 cfu/ml.
Fig. (2): Mortality of Culex pipiens 3rd instar larvae
treated with Bacillus thuringiensis isolates for
different times at the concentration of 109 cfu/ml.
Fig. (3): Mortality of Culex pipiens 3rd instar larvae treated with Bacillus thuringiensis isolates and the
commercial biopesticide Wertobag for different times at the concentrations of 109 cfu/ml and 32000,
to the larvae of the C. pipiens. Bt can be isolated from
soil, leaves, dead larvae or water (Armengol et al.,
2007; Hernández-Soto et al., 2009; Liang et al., 2011
and Valicente et al., 2010). The Bt strain produces
crystal proteins that have been successfully used for
controlling the mosquito population (Liang et al.,
Bacillus was confirmed morphologically and
biochemically accordingly to Sneath (1986). The
isolates were subjected to further biochemical
characterization test according to Claus and Berkeley
(1986) and resulted in four isolates closely
resembling B. thuringiensis 22.19. Wild strains
isolated form environmental samples can synthesize
crystals that display higher activity against insect
pests in comparison to Bt strains already used in
pesticide production. Previous workers were not able
to isolate this entomopathogenic bacterium from
different habitats in black sea region including
hazelnut orchards. Results showed that Bt22.19 is an
important strain that produces secondary metabolites
and active compounds. The strain plays an important
role in biological control of C. pipiens larvae.
It is well known that the characteristic shape of the
mosquitocidal crystals is the spherical-shaped
crystals. Bt produces this type of crystals (Charles and
de Barjac, 1982), to which Culex larvae are more
susceptible (Boisvert, 2005). In the present study, the
highest mortality percentage recorded was 100%,
obtained by the isolate Bt22.19, as compared to other
strains in the untreated control. Present results
reported that successful pupation of Culex spp. was
significantly delayed when exposed to Bt22.19. In
addition, Lacy et al. (2004) confirmed that Bt strains
affected C. quinquefasciatus.
Larvicidal effects of Bt isolates and its commercial
biopesticide Wertobag were compared using
percentages of mortality of C. pipiens larvae at 6, 12,
24 and 48h post treatment. The results showed a good
performance for the isolate of Bt22.19 (100%
mortality at a concentration of 109 cfu/ml within 48h)
and the commercial biopesticide Wertobag (100%
mortality at concentrations of 16000 and 32000
ITU/mg within 48h). Isolate Bt22.19 that had higher
activity than the other isolates (Btbmeg, Bthma7 and
Bthma5) against C. pipiens larvae were identified in
spite of their high similarity cry genes. These data
support the idea that although a great variability in cry
genes codifying for different mosquitocidal toxins
that exist in the natural strains of B. thuringiensis
(Schnepf et al., 1998). Similarly, the results suggest
that insecticidal potency of the isolates was not
directly related with their cry gene content as stated
by Padidam (1992). In addition, Seifinejad et al.
(2008) confirmed that presence of specific genes was
not an accurate indicator of toxicity, because the
genes could be inactive, under the control of an
inefficient promoter or be expressed in a
concentration too low to affect toxicity. However, the
detection of these genes in most Bt isolates collected
locally indicated that they can be effectively used to
produce spore-crystal mixture for controlling
mosquitoes. Similarly, many studies stated that Cry4
and Cry10 play a major role in mosquitocidal activity
of Bt strains (Delecluse et al., 1988; Guerchicoff et
al., 1997; Ben-Dov et al., 1999; Armengol et al.,
2007; Hernández-Soto et al., 2009 and Baig and
Mehnaz, 2010). Misztel et al. (1996) reported that
differences in potency, in general, could be attributed
to the differences in susceptibility of the treated
insects, where explained the connection between
insect mortality and exposure time. This study
reported that highly susceptible insects stopped
feeding within 1 hour and died within 12-48h after
ingestion of the toxin, less susceptible ones ceased
feeding after 6 h and died after 2 days. While the
slightly susceptible insects stopped feeding after 24 h
and died after 2 weeks.
In conclusion, larvicidal potency of the two novel
crystal protein genes, cry4A and cry10, was encoded
at highly mosquitocidal Bt isolate Bt22.19. It is
essential to perform additional bioassays with these
Cry toxins against resistant mosquito colonies
selected ith B.thuringiensis subsp. israelensis toxins.
Bt22.19 exhibited an effect effortlessly against
C. pipiens larvae. So, it can be used as an alternative
insecticide because it is safe for the environment.
Further studies are needed to identify the active
compounds that can be used in broad spectrum for
controlling insects and also to determine the mode of
action of these compounds.
Thanks are due to TUBITAK, the scientific and
technological research council of Turkey that funded
this research.
Almeida, A. P., R. P. Galão, C. A. Sousa, M. T. Novo,
R. Parreira, and J. Pinto 2008. Potential mosquito
vectors of arbo-viruses in Portugal: species,
distribution, abundance and West Nile infection.
Trans. R. Soc. Trop. Med. Hyg. 102(8):823-32.
Armengol, G., M. C. Escobar, M. E. Maldonado, and
S. Orduz 2007. Diversity of Colombian strains of
Bacillus thuringiensis with insecticidal activity
against dipteran and lepidopteran insects. J. Appl.
Microbiol. 102(1): 77-88.
Baig, D. N. and S. Mehnaz 2010. Determination
and distribution of cry-type genes in
halophilc Bacillus thuringiensis isolates of
Arabian Sea sedimentary rocks. Microbiol. Res.
165(5): 376-383.
Ben-Dov, E., G. Nissan, N. Pelleg, R. Manasherob, S.
Boussiba, and A. Zaritsky 1999. Refined, circular
restriction map of the Bacillus thuringiensis
subsp. israelensis plasmid carrying the mosquito
larvicidal genes. Plasmid 42:186191.
Boisvert, M. 2005. Utilization of Bacillus
thuringiensis var. israelensis (Bti)-based
formulation for the biological control of mosquito
in Canada. Pp. 87-93 In: J. L. Cote, I. S. Otvos, J.
I. Schwartz, and C. Vincent (eds). 6th Pacific Rim
Con. Biotech. of Bacillus thuringiensis and its
Environmental Impacts. Fairmont Empress Hotel,
Victoria, BC, Canada, Oct. 30- Nov. 3, 2005.
Charles, J. F., and H. de Barjac 1982. Sporulation et
cristallogénèse de Bacillus thuringiensis var.
israelensis en microscopie électronique. Ann. Inst.
Pasteur Mic. 133:425-442.
Claus, D., and C. W. Berkeley 1986. The genus
Bacillus. In: Bergey’s Manual of Systematic
Bacteriology. Vol 2. Sneath, P.H.A. (Ed).
Williams, Wilkins, Baltimore. 34: 1105-1139.
Delecluse, A., C. Bourgouin, A. Klier and G.
Rapoport 1988. Specificity of action on mosquito
larvae of Bacillus thuringiensis var. israelensis
toxins encoded by two different genes. Mol. Gen.
Genet. 214:42-47.
Gill, S. S., E. A. Cowles, and P. V. Pietrantonio 1992.
The mode of action of Bacillus thuringiensis
endotoxins. Annu. Rev. Entomol. 37:615-636.
Guerchicoff, A., R. A. Ugalde, and C. P. Rubinstein
1997. Identification and characterization of a
previously undescribed cyt gene in Bacillus
thuringiensis subsp. israelensis. Appl. Environ.
Microbiol. 63:27162721.
Harbach, R. E., C. Dahl, and G. B. White 1985. Culex
(Culex) pipiens Linnaeus (Diptera: Culicidae):
concepts, type designations and descriptions.
Proc. Entomol. Soc. Wash. 87: 124.
Harbach, R. E. 2012. Culex pipiens: species versus
species complex taxonomic history and
perspective. J. Am. Mosq. Contr. Assoc. 28:10-23.
Hernández-Soto, A., M. C. Del Rincon-Castro, A. M.
Espinoza, J. E. Ibarra 2009. Parasporal body
formation via over expression of the Cry10Aa
toxin of Bacillus thuringiensis subsp. israelensis,
and Cry10Aa-Cyt1Aa synergism. Appl. Environ.
Microbiol. 75:4661-4667.
Höfte, H. and H. R. Whiteley 1989. Insecticidal
crystal protein of Bacillus thuringiensis.
Microbiol. Rev. 53(2):242-255.
Ichimatsu, T., E. Mizuki, K. Nishimura, T. Akao, H.
Saitoh, and K. Higuchi 2000. Occurrence of
Bacillus thuringiensis in fresh waters of Japan.
Curr. Microbiol. 40(4): 217-220.
Iriarte, J., M. Porcar, M. Lecadet, and P. Caballero
2000. Isolation and characterization of Bacillus
thuringiensis strains from aquatic environments in
Spain. Curr. Microbiol. 40:402-408.
Lacy, L. A., J. Day, and M. Heitzman 2004. Long
term effects of Bacillus sphaericus on Culex
quinfasciatus. J. Inverteb. Pathol. 49(1):116-123.
Liang, H., Y. Liu, J. Zhu, P. Guan, and S. Li 2011.
Characterization of cry2-type genes of Bacillus
thuringiensis strains from soil-isolated of Sichuan
basin, China. Braz. J. Microbiol. 42, 140-146.
Misztel, L., W. G. Musial, and J. Augustyniak 1996.
Insecticidal toxins of Bacillus thuringiensis. Post
Mikrobiol. 25: 193-211.
Onder, H. 2007. Using permutation tests to reduce
type I and II errors for small ruminant research. J.
Appl. Anim. Res. 32(1):69-72.
Padidam, M. 1992. The insecticidal crystal protein
CryIA(c) from Bacillus thuringiensis is highly
toxic for Heliothis armigera. J. Invertebr. Pathol.
Regis, L., M. H. Silva-Filha, C. Nielsen-Leroux, and
J. F. Charles 2001. Bacterial larvicides of dipteran
disease vectors. Trends. Parasitol. 17:377-380.
Roh, J. Y., J. Y. Choi, M. S. Li, B. R. Jin, and Y. H.
Je 2007. Bacillus thuringiensis as a specific, safe,
and effective tool for insect pest control. J.
Microbiol. Biotechnol. 17(4):547559.
Salehi, J. G., A. A. Pourjan, A. Seifinejad, R.
Marzban, K. Kariman, and B. Maleki 2008.
Distribution and diversity of Dipteran-specific cry
and cyt genes in native Bacillus thuringiensis
strains obtained from different ecosystems of Iran.
J. Ind. Microbiol. Biotechnol. 35(2):83-94.
Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus,
J. Baum, J. Feitelson, D. R. Zeigler, and D. H.
Dean 1998. Bacillus thuringiensis and its
pesticidal crystal proteins. Microbiol. Mol. Biol.
Rev. 62(3):775-806.
Seifinejad, A., G. R. S. Jouzani, A. Hosseinzadeh, and
C. Abdmishani 2008. Characterization of
Lepidoptera-active cry and vip genes in Iranian
Bacillus thuringiensis strain collection. Biol.
Control. 44:216-226
Sneath, P. A., N. S. Mair, M. E. Sharpe, and J. G.
Holt. 1984. Bergey’s manual of systematic
bacteriology, (2):1123, Williams & Wilkins,
Sneath, P. H. A. 1986. Endospore-forming gram
positive Rods and Cocci. p. 1104-1105 In: Sneath,
P.H.A., N.S. Mour, M.E. Sharpe, and J.G. Holt
(eds). Bergy's Manual of Systematic Bacteriology,
8th edition. Vol. 2, William & Wilkins. Baltimore.
Swiecicka, I., K. Fiedoruk, and G. H. Bednarz 2002.
The occurrence and properties of Bacillus
thuringiensis isolated from free- living animals.
Lett. Appl. Microbiol. 34:194-8.
Valicente, F. H., A. T. P. Edgard, J. V. V. Maria, P.
C. Newton, A. C. Andréia, T. G. Cláudia, and G.
L. Ubiraci 2010. Molecular characterization and
distribution of Bacillus thuringiensis cry1 genes
from Brazilian strains effective against the fall
armyworm, Spodoptera frugiperda. Biol. Control.
53: 360-366.
WHO. 2005. Guidelines for laboratory and field
testing of mosquito larvicides.World Health
Organization, Geneva, Switzerland.
Yu, Y. M., M. Ohba, and S. S. Gill
1991. Characterization of mosquitocidal activity
of Bacillus thuringiensis subsp. fukuokaensis
crystal proteins. Appl. Environ. Microbiol.
... The chemical methods has to many side effect to natural and human body. For this reason, biological control as natural methods has been commonly used for plant protection [22,23]. As well as integrated pest management (IPM) programs [4,10] to combat them in other areas [2,12]. ...
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Beauveria bassiana is a fungi which used as biological control factor against Sesamia cretica Led. S. cretica is usually only one component of a complex of stem borers attacking corn plants. Chemical control is of limited use. Because the chemical control is heavy risk for nature. And the general the insect is towards the use of non-chemical methods, such as biological control. The efficincy of two isolates of B. bassiana (1,2); with some adjuvants (dispersive, carriers) added to the suspended solution of the fungi in soy bean extract and date syrup. Results revealed that two isolates reduced the percentage of the corn plant infection as compared with control treatment, it was also indicated that spraying corn plants by the suspension of the isolate with DMS (0,02 %) was significantly reduced the percentage of infestation to (1.62 %) compared with control treatment (21.86 %) after (36) days of treatment. Whereas the results of studying the efficiency of different cultural medium for propagation of B. bassiana showed that the soya bean extract alone and combined with date syrup achieved the best mycelium growth and sporulation after seven days from culture compared either other culture medium as it reached 264.3 mg, 243.6 mg and 217.5 *10 6 , 252.5×10 6 spore each 0.1gm, respectively.
... One control considered as safe is to use bioinsecticides. One bacteria Bacillus thuringiensis(Bt) is well-known for this purpose 3 . ...
Nine strains belonging to the Bacillus genus have been isolated from soil samples in Tien Phuoc district, Quang Nam province. They were capable of surviving at 42°C, and the Bl, B4, B7 and B9 strains could produce toxic crystals at this temperature. B7 had the most promising characteristics in terms of spore-forming ability. The result of the 16S rRNA gene sequencing showed that B7 belongs to the Bacillus thuringiensis species, which is known to effectively control root-knot Meloidogyne sp. nematodes attacking pepper tree. This study was aimed at evaluating the inhibitory effect of the bacterial isolate under study on root-knot eggs and juveniles. It was shown that the highest inhibitory activity of the cultures of the bacterial strains under study was observed at their concentration of 109cells/mL; in this case, up to 89.67% of nematode eggs and 100% of juveniles J2 were killed after 10 h of treatment. Bacillus thuringiensis, Meloidogyne sp., pepper tree, root-knot, nematodes We are grateful to the Danang University of Science and Education and Duy Tan University for supporting this study.
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Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%), followed by cry2Aa (6.8%) and cry2Ab alone (2.5%), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.
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The history of the concept of Culex pipien.s Linnaeus is reviewed. An illustration of a syntype published by Reaumur is designated as the lectotype of pipiens. A lectotype is also designated for Culex bifhcatus Linnaeus, which is stabilized as a synonym of pipiens. A neotype for pipiens is designated in place of the non-extant lectotype-specimen. The adult, pupal, and larval stages of the neotype are described and illustrated. Sexual differences are described and ilIus- trated for the alloneotype. A description of diagnostic and variable characters is provided for each life stage of the species. The present system of naming organisms originated with the work of Carolus Linnaeus, and the 10th edition of his Systema Naturae (1758) is the designated
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The taxonomic history of Culex pipiens (1758-present) is reviewed. The central question is whether Cx. pipiens is a single polytypic species or a complex of sibling species? The taxon traditionally known as the Cx. pipiens complex is referred to as the Pipiens Assemblage to avoid difficulties associated with the meaning of the word "complex". Neotype specimens have been designated to fix the morphological identities of Cx. pipiens, Cx. molestus, and Cx. quinquefasciatus, and Cx. pallens is represented by a holotype, but whether these nominal forms represent one or more biological species remains controversial. Despite extensive morphological and physiological/behavioral variation, there is no indication of subspecific or racial differences in geographically separated populations of Cx. pipiens. Introgression occurs where populations of Cx. pipiens and Cx. quinquefasciatus overlap, but the retention of parental epiphenotypes outside the zone of introgression provides evidence of independent species cohesion. The main conclusions reached are: Cx. pipiens and Cx. quinquefasciatus are separate species which evolved in Africa and hybridize in non-indigenous areas where they were unintentionally introduced by humans; Cx. molestus is nothing more than a phenotypic and physiological variant of Cx. pipiens; and Cx. pallens has no taxonomic status under the provisions of the International Code of Zoological Nomenclature. Based on morphological similarity, the Pipiens Assemblage includes Cx. pipiens, Cx. quinquefasciatus, and possibly Cx. australicus. There is no evidence to suggest that the Pipiens Assemblage includes any other species.
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Önder, H. 2007. Using permutation tests to reduce type I and II errors for small ruminant research. J. Appl. Anim. Res., 32: 69–72.Although parametric tests (F or t) are considerably effective, these are sometimes ineffective when the assumptions needed by model are not provided. In such a case, permutation test unaffected by the assumptions can be applied as a non-parametric method. It has been observed by citing an example that permutation test produces more reliable results than one-way ANOVA in terms of type I error rate and power of the test and permutation test is recommended in order to avoid type I and II errors and to prevent the potential profit lost.
All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.
The characterization of 70 Bacillus thuringiensis (Bt) strains isolated from different agro-ecological regions of Iran is presented. Characterization was based on PCR analysis using 25 general and specific primers for cry1, cry2, cry9 and vip3Aa genes encoding proteins active against Lepidoptera, crystal morphology, plasmid profiles, and protein band patterns as well as their insecticidal activity on Heliotis armigera. Isolates containing vip3Aa gene were the most abundant (82.6%) followed by those containing cry2 (56.5%), cry1 (49%) and cry9 (30%). Twenty-two distinct cry1-type profiles were identified from only cry1-harboring isolates when these were analyzed with specific primers. Several of them were found to be different from all previously published profiles. Finally 7.24% of the isolates did not produce any PCR product. Some strains were positive by universal primers but negative by specific primers for all known genes of cry1, cry2 and cry9 or gave PCR products of different sizes when assayed with cry1C, cry1E, cry1J, cry9A, and vip3a specific primers. These strains may contain a new gene or genes that seem promising for biological control of insects and management of resistance. Based on morphological and molecular studies, 20 potentially Lepidopteran-specific active isolates were selected for bioassays. Four strains showed similar or higher activity against H. armigera larvae than Bt subsp. kurstaki (Btk) and displayed high similarities with the Btk used in this study with regard to protein and plasmid profiles and the presence of cry genes. These results are important for directing future exploration of microbial control strategies for control of crop pests in the region.