Microbiological Research 168 (2013) 512517

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis

strain B-JJX with specic toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae
Yuan Zhang a , Guiling Zheng a , Jianxin Tan b , Changyou Li a, , Linyou Cheng a,c
a
b
c

Key Lab of Integrated Crop Pest Management of Shandong Province, College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China
College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, China
Shandong Institute of Biopharmaceuticals, Jinan 250101, China

a r t i c l e

i n f o

Article history:
Received 28 January 2013
Received in revised form 26 February 2013
Accepted 2 March 2013
Available online 28 March 2013
Keywords:
Biological control
cry8 gene
Scarabaeid larvae
Insecticidal activity
PCR-RFLP

a b s t r a c t
Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with
specic toxicity is of great importance to biological control of insect pests. In this study, by screening
66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene
from Bt strain B-JJX was identied via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the
Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73 . The open reading frame of the
cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids
with a putative MW of 133.3 kDa which was conrmed by SDS-PAGE analysis. The Cry8Ab1 protein was
expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73
along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid
pests, Holotrichia oblita and H. parallela, with an LC50 of 5.72 and 2.00 g toxin g1 soil, respectively. The
results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC50
of 1.72 and 0.96 g toxin g1 soil against H. oblita and H. parallela, respectively.
2013 Elsevier GmbH. All rights reserved.

1. Introduction
Grubs, larvae of scarabaeid beetles (Coleoptera: Scarabaeidae),
are common pests that can cause extensive damage to crops and
plants including grain, cotton, oil crops, vegetables, tobacco, grass
and other plants (Bohlen and Barrett 1990; Schnetter et al. 1996;
Silva et al. 2010), and are ranked rst in all types of soil pests
in China (Wei et al. 1995; Luo et al. 2008). Because grubs live in
subterranean environments and to some extent are able to avoid
the effect of chemicals, grub control by chemicals usually is not as
efcient as expected (Adams et al. 2002; Koppenhofer et al. 2000;
Mannion et al. 2001). On the other hand, chemical pest control may
pose risks via chemical residues to the environment and human and
animal health (Liesch and Williamson 2010; Redmond and Potter
2010). Therefore, nding safe and effective preventive approaches
to control grubs is a national priority.
Bacillus thuringiensis (Bt), a species of Bacillus including many
subspecies, can produce crystals which have high toxicity to a variety of insects (Sanahuja et al. 2011). Due to the features of Bt,
such as high insecticidal activity, relative specicity, and safety

Corresponding author at: Qingdao Agricultural University, Changcheng Road,

Chengyang District, Qingdao 266109, China. Tel.: +86 532 86080462;
fax: +86 532 86080447.
E-mail addresses: cyli@qau.edu.cn, changyouli99@yahoo.com (C. Li).
0944-5013/$ see front matter 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.micres.2013.03.003

to human, animals, it has been extensively applied to the control

of agricultural pests, forest pests, warehouse and sanitation pests
(Schnepf et al. 1998). Before the 1980s, most research focused on
lepidopteran and dipteran-specic isolates/strains (Goldberg and
Margalit 1977; Schnepf et al. 1998). The rst Bt strain with the
toxicity specic to coleopteran pests, Tenebrio molitor and Leptinotarsa decemlineata was isolated in 1983 (Krieg et al. 1983). However,
only relatively few studies were devoted to grub control with Bt.
Ohba et al. (1992) isolated B. thuringiensis serovar japonensis strain
Buibui, which has specic toxicity to scarabaeid larvae. This strain
was used to control Anomala cuprea by Suzuki et al. (1992). Subsequently, more than 20 new Bt strains with specic insecticidal
activity to scarabaeid larvae have also been isolated (Yu et al. 2006;
Shu et al. 2009b; Crickmore 2013). Cloning and characterization
of cry gene with activity to grubs is becoming more important in
the eld of pest control. Sato et al. (1994) cloned cry8Ca1 gene
from Bt strain Buibui with activity to A. cuprea. The cry8Aa1 and
cry8Ba1 gene, with high insecticidal activity to many species of
scarabaeid larvae, have been cloned from the B. thuringiensis subsp.
kumamotoensis (Michaels et al. 1996). In recent years, cry8Ca2,
cry8Ea1, cry8Ea2, cry8Fa1, cry8Ga1 and cry8Ha1 genes with activities against scarabaeid larvae have also been cloned in China (Shu
et al. 2007, 2009a,b). Currently, totally fouty-eight cry8-type genes
with specic toxicity to different species of scarabaeid larvae have
been cloned (Crickmore 2013). In this study, we cloned a novel
cry8 insecticidal protein gene from Bt strain B-JJX isolated by our

Y. Zhang et al. / Microbiological Research 168 (2013) 512517

laboratory. This strain had insecticidal activity to a variety of

scarabaeid pests, and we sought to improve its efciency by transforming the cry8Ab1 gene into Bt acrystalliferous mutant strain
HD-73 . The expression of cry8Ab1 gene in HD-73 and its insecticidal activity were investigated in this study. The ndings will lay
a foundation for grubs control through providing the new cry gene
for construction of genetically engineered microbes or engineered
insecticidal crop.
2. Materials and methods
2.1. Strains, plasmids, insects and culture media
The Bt strain B-JJX was isolated from soil samples collected near
Yantai, Shandong Province, Eastern China, in 2006 using a thermal
shock treatment (Zhang et al. 2000). The strain was deposited in
China Center for Type Culture Collection (CCTCC), Wuhan, China
(Collection number: CCTCC AB 2013034). The Bt acrystalliferous
mutant strain HD-73 , Escherichia coli SSC110 and the Bt expression
vector pSXY422b were generously provided by Dr. Fu-ping Song,
Institute of Plant Protection, Chinese Academy of Agricultural Sciences. E. coli DH5 and clone vector pBluescript SK(+) were stored
in this lab. Bt and E. coli were cultured in LB medium at 28 C and
37 C, respectively.
Two Asian species of scarabaeid, Holotrichia oblita and
Holotrichia parallela were collected from the eld in Chengyang
District, Qingdao, China. The adults were fed with willow leaves
and kept indoors at 25 C until they laid eggs. The emerging larvae
were fed with potato for 3 days for bioassay. A coleopteran species,
T. molitor, and three lepidopteran species, Helicoverpa armigera,
Spodoptera exigua, Mythimna separata were incubated at 26 C, 85%
RH and a photoperiod of 16:8 (L:D) in this lab, the neonate larvae
were used for bioassay.
2.2. PCR-RFLP analysis of B. thuringiensis strains
Plasmid DNA extracted from 66 B. thuringiensis strains isolated
from soil samples collected in different areas of Shandong Province,
China, was used as template for polymerase chain reaction (PCR)
amplication. The Bt plasmid DNA extraction method was followed
the protocol described by Shu et al. (2009b). Identication of cry8type genes was performed using PCR-RFLP method established by
Yu et al. (2006).
2.3. Cloning and characterization of the cry8Ab1 gene
To amplify the cry8 gene from Bt strain B-JJX, a
pair of specic primers, a forward primer JJX5 (5 CGGGATCCGATGAGTCCAAATAAT-3 ) and a reverse primer JJX3
(5 -GCGTCGACTTACTCTACGTC-3 ) (letters with underline indicate cutting site of BamHI or SalI, respectively), were designed
with software Primer Premier 5.0 based on the sequence of the
cry8-type genes cited in GenBank. Primers were synthesized by
Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Taq DNA
polymerase and dNTPs were purchased from TransGen Biotech
Co., Ltd. (Beijing, China). PCR was performed using plasmid DNA
as template isolated from Bt strain B-JJX. The PCR program was as
follows: 94 C for 5 min and 30 cycles of denaturation at 94 C for
1 min, annealing at 54 C for 1 min and extension at 72 C for 4 min
followed by a 10-min nal extension at 72 C. An approximate
3.5 kb long PCR product, conrmed by sequencing, was ligated
with pBluescript SK(+) to construct the recombinant pBlue-cry8.
The recombinant plasmid pBlue-cry8 was extracted and analysis
by digestion with BamHI and SalI after amplied in E. coli DH5.
Restriction enzymes were purchased from TaKaRa Company
(Dalian, China). The methods for E. coli plasmid extraction, DNA

513

digestion, ligation, and transformation were followed the protocols

of Sambrook et al. (1989). DNA sequencing was carried out by the
United Gene Company (Shanghai, China).
The amino acid sequences of the Cry8-type proteins and Cry1Aa
protein were downloaded from GenBank and edited using Bioedit
software. Then a dendrogram was generated using MEGA5 software.
2.4. Expression of the cry8Ab1 gene in B. thuringiensis
acrystalliferous mutant strain
The ORF of cry8Ab1 gene prepared from pBlue-cry8 digested
with BamHI and SalI was inserted into an expression vector
pSXY422b to construct a recombinant expression vector pSXYcry8. Then pSXY-cry8 was transformed into E. coli SCS110 to
demethylated, and then electroporated into Bt crystal mutant HD73 followed the method of Lereclus et al. (1989). A positive clone,
HD73-cry8Ab1, was identied by PCR and then cultured on LB
agar plates containing 25 g/mL of erythromycin at 28 C until the
spores formed and released. The cells, spores and parasporal crystals were collected and prepared for scanning electron microscopic
observation and SDS-PAGE analysis.
For scanning electron microscopic observation, after autolysis,
the spore-crystal complex of each strain (HD-73 , HD73-cry8Ab1
and Bt B-JJX) was collected by centrifugation at 10,000 r/min for
10 min at 4 C. The pellets were resuspended in sterile-distilled
water. Sample preparation and scanning electron microscopic
observation (JEOL JSM-7500F, Japan) was performed following the
procedure in Xue et al. (2008).
For SDS-PAGE analysis, samples extracts from HD-73 , HD73cry8Ab1 and Bt B-JJX were washed once in 1 mol/L NaCl and once
in distilled water and then resuspended in sample buffer (mixed
equal volume of sample with 2SDS sample buffer). Samples were
analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained by Coomassie blue R-250
(Sambrook et al. 1989). Protein molecular weight standards and
DNA molecular weight standards were purchased from TransGen
Biotech Co., Ltd. (Beijing, China).
2.5. Bioassay of insecticidal activity of the cry8Ab1 gene
Bt strains HD-73 , HD73-cry8Ab1 and Bt B-JJX were cultured
in LB medium until spores and parasporal crystals were released.
Cells, spores and parasporal crystals were prepared by centrifugation at 10,000 r/min for 10 min. Protein concentration in samples
were analyzed using Bradford method (Bradford 1976). The insecticidal activity of Bt strain B-JJX, HD73-cry8Ab1, HD-73 against H.
oblita and H. parallela was performed according to the method of
Yu et al. (2006). The insecticidal toxicity of Bt strain B-JJX, HD73cry8Ab1, HD-73 against H. armigera, S. exigua, M. separata, T.
molitor was performed according to the method of Xue et al. (2005).
Each bioassay was repeated at least three times. Bioassay data were
analyzed using the DPS data processing system (Tang and Zhang
2012).
3. Results
3.1. Screening of B. thuringiensis strains containing cry8-type
gene using PCR-RFLP
Sixty-six B. thuringiensis strains isolated from soil samples collected from Shandong Province of China were screened by PCR
method with the universal primers S5un8/S3un8 which were
applied for identication of cry8-type genes. A single 1.2 kb-long
fragment was amplied using DNA template isolated from Bt strain
B-JJX (data not shown), indicating that the Bt strain B-JJX contained

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Y. Zhang et al. / Microbiological Research 168 (2013) 512517

Fig. 1. Construction of the recombinant plasmids pBlue-cry8 and pSXY-cry8. (A) Flowchart for construction of the recombinant plasmids pBlue-cry8 and pSXY-cry8. (B)
Restriction digestion analysis of the recombinant plasmid pBlue-cry8. Lane 1: -HindIII DNA Marker (23130, 9416, 6557, 4361, 2322, 2027, 564 bp); lane 2: pBlue-cry8
digested with BamHI; lane 3: pBlue-cry8 digested with SalI; lane 4: pBlue-cry8 digested with BamHI and SalI; lane 5: PCR product of cry8 gene. (C) Restriction digestion
analysis of the recombinant plasmid pSXY-cry8. Lane 1: -HindIII DNA Marker (23130, 9416, 6557, 4361, 2322, 2027, 564 bp); lane 2: pSXY-cry8 digested with BamHI and
SalI; lane 3: plasmid pSXY422b digested with BamHI and SalI; lane 4: PCR product of cry8 gene.

Y. Zhang et al. / Microbiological Research 168 (2013) 512517

515

Fig. 2. Diagram of the dendrogram of Cry8-type protein and Cry1Aa.

a cry8-type gene which may encode a Cry8 protein potentially

with insecticidal activity against scarabaeid larvae. The 1.2 kb-long
PCR product could not be digested by DraI and EcoO109I (data not
shown) and this PCR-RFLP pattern differed from the known cry8
genes, suggesting that B-JJX may contain a novel cry8-type gene.
The morphological observation of Bt strain B-JJX showed its cells
were single rods or formed a chain. After cultivated on LB plates at
28 C for 3 days, it produced the long oval spores and the spherical
or cuboidal parasporal crystals in different sizes (Fig. 3A).
3.2. Cloning and characterization of the cry8Ab1 gene
A 3.5 kb-long PCR product corresponding to the open reading
frame of the cry8 gene was amplied using a pair of specic primers,
JJX5 and JJX3, designed according to cry8-type genes. This fragment
was inserted into a cloning vector pBluescript SK(+) resulting in
generation of the recombinant plasmid pBlue-cry8 (Fig. 1A). Subsequently, pBlue-cry8 was identied by PCR and double restriction
digestion with BamHI and SalI (Fig. 1B), which proved that the target cry8 gene was successfully inserted into pBluescript SK(+). The
cry8 gene consisted of 3543 bps with a G + C content of 37.99% and
encoded a protein of 1180 amino acids. The deduced molecular
weight (MW) of Cry8 was 133.3 kDa with an isoelectric point of pH
4.68 indicating a weakly acidic protein. The amino acid sequence
of the cry8 gene shares the highest homology (75%) with that of
Cry8Aa1 and <75% with that of the other cry8 genes. Comparing the
amino acid sequence of Cry8Ab with other Cry8 ICPs and Cry1Aa, a
dendrogram of the Cry8 proteins was generated (Fig. 2). As shown
in Fig. 2, all Cry8-type ICPs clustered to a group. It consisted of several sub-branches at different level and forms a higher level branch
with Cry1Aa. Cry8Ab and Cry8Aa were in the same clade that indicated they shared more conservative amino acid sequences and had
a close genetic relationship in comparison with the other Cry8-type
ICPs.
The sequence of the cry8 gene was registered in GenBank (Accession number is EU044830), and named cry8Ab1 as a novel cry gene
by the B. thuringiensis delta-endotoxin nomenclature committee

Fig. 3. Morphology of parasporal crystals and spores from the Bt strain B-JJX (A),
the engineered Bt strain HD73-cry8Ab1 (B) and the Bt acrystalliferous mutant strain
HD-73 (C). Bar = 1 m.

(Crickmore 2013). According to the principle of nomenclature for

cry gene (Crickmore et al. 1998), the last Arabic number 1 in
cry8Ab1 meant that cry8Ab1 was the rst one which was submitted
to the nomenclature committee and got their approved among the
cry8Ab genes.

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Y. Zhang et al. / Microbiological Research 168 (2013) 512517

Table 1
Bioassay results of the Cry8Ab1 protein and the Bt strain B-JJX to Holotrichia oblita and H. parallela.
Bt strain or protein

Insect species

Regression equation

Determination coefcient (r2 )

LC50 (95% CI) (g toxin g1 soil)

Cry8Ab1

Holotrichia oblita
Holotrichia parallela
Holotrichia oblita
Holotrichia parallela

Y = 4.29 + 0.93X
Y = 4.55 + 1.49X
Y = 4.70 + 1.29X
Y = 5.03 + 1.45X

0.9955
0.9005
0.9302
0.9750

5.72 (1.3110.20)
2.00 (0.663.50)
1.72 (0.782.63)
0.96 (0.291.63)

B-JJX

T. molitor, and the three lepidopteran larvae, H. armigera, S. exigua,

and M. separata at the concentration of 500 g toxin g1 diet.
4. Discussion

Fig. 4. SDS-PAGE analysis of parasporal crystal protein from Bt strains. Lane 1:

protein marker (200, 116, 97.2, 66.4, 44.3 kDa); lane 2: engineered Bt strain HD73cry8Ab1; lane 3: Bt strain B-JJX; lane 4: Bt acrystalliferous mutant strain HD-73 .

3.3. Expression of cry8Ab1 gene in the Bt acrystalliferous mutant

strain HD-73
The DNA fragment containing cry8Ab1 gene, digested with
BamHI and SalI was inserted in the Bt expression vector pSXY422b
resulting in the construction of a recombinant plasmid, pSXY-cry8
(Fig. 1A). After identifying by double digestion analysis with BamHI
and SalI (Fig. 1C), pSXY-cry8 was transformed into the Bt acrystalliferous mutant strain HD-73 . A positive clone, HD73-cry8Ab1,
was cultured for 72 h until the spores formed and were released.
The morphology of crystal of Cry8Ab1, observed with a scanning
electron microscope, showed spherical parasporal shape (Fig. 3B)
which was similar to the spherical ones formed by the wild-type
Bt strain B-JJX (Fig. 3A). HD-73 did not form any parasporal crystals (Fig. 3C). SDS-PAGE analysis indicated that the cry8Ab1 gene
expressed an approximately 130 kDa (MW) protein (Fig. 4), which
was consistent with the prediction. There was no 130 kDa protein
in the original HD-73 .

3.4. Insecticidal activity of the Cry8Ab1 protein and the Bt strain

B-JJX
The Cry8Ab1 protein was toxic to the 3-day-old larvae of
H. oblita and H. parallela with LC50 of 5.72 g toxin g1 soil and
2.00 g toxin g1 soil, respectively. The Bt strain B-JJX was also toxic
to the 3-day-old larvae of both H. oblita and H. parallela with an
LC50 of 1.72 g toxin g1 soil and 0.96 g toxin g1 soil, respectively
(Table 1). On the contrary, HD-73 did not show any insecticidal
activity to the scarabaeid larvae. The Bt strain B-JJX and the Cry8Ab1
protein had no insecticidal activities against the coleopteran larvae,

Different insecticidal crystal protein (ICP) produced by B.

thuringiensis have the insecticidal activity to diverse pests
(Sanahuja et al. 2011). The toxic specicity of ICP is closely associated with the type of cry genes (Schnepf et al. 1998). The
identication of the type of cry gene may help to presume the
insecticidal efciency and/or specicity of a certain ICP encoded
by the gene (Kuo and Chak 1996; Noguera and Ibarra 2010). In
this study, we rst demonstrated that the Bt strain B-JJX contained a novel cry8 gene (cry8Ab1), which was subsequently cloned
and expressed in Bt acrystalliferous mutant strain HD-73 . The
known cry8 genes have specic toxicity to scarabaeid grubs (Shu
et al. 2009b; Crickmore 2013). We veried the expected insecticidal specicity of B-JJX or HD73-cry8Ab1 by testing its efciency
against H. oblita and H. parallela, which are the main scarabaeid
pests in China. We demonstrated the usefulness of the PCR-RFLP
method for both identication of cry genes and a prediction of the
insecticidal activity of its encoding ICP. Since bioassays are time
consuming and complicated, the application of PCR-RFLP assays
could shorten and simplify the preliminary screening. Moreover,
the PCR-RFLP method also can help to nd novel cry genes based
on the PCR-RFLP pattern of the tested cry gene.
The family Scarabaeidae as currently dened consists of over
30,000 species of beetles worldwide. The dominant species of this
family are different from each other in different areas around the
world. Therefore, currently there is no uniform standard method
for testing insecticidal activity of Cry ICPs to grubs. The dominant
species of the family Scarabaeidae in Shandong province of China
are H. oblita and H. parallela. Up to now, Yu et al. (2006) and Shu
et al. (2009a,b) have reported that the Bt strain BT185 (cry8Ea)
and the Bt strain HBF-18 (cry8Ga) are toxic to H. parallela. As to
the toxin against H. oblita, only the Bt strain HBF-18 (cry8Ga) was
reported (Shu et al. 2009b). Our bioassay data indicated that we
have found a new Bt strain B-JJX with the cry8Ab1 gene which have
high insecticidal activity against two scarabaeid larvae, H. oblita
and H. parallela. However, bioassay results also show the insecticidal activity of Cry8Ab1 expressed by HD-73 is lower than that
of B-JJX. This phenomenon may occur because the Bt acrystalliferous mutant strain HD-73 may produce less Cry8Ab1 protein.
The other possible factor is that the wild strain B-JJX produces two
types of crystals, spherical and cuboidal, of different size. One or
both of them may contain other toxin(s) in addition to Cry8Ab1,
which needs further investigation.
More than 40 cry8-type ICP genes are known world-wide
(Crickmore 2013). Some of them have been developed as bioinsecticides or transformed into plants (Sato et al. 1994; Shu et al. 2007;
Liu et al. 2010). These facts display a great potential and a broad
application prospect of Bt strains and its cry8 genes in the biocontrol of insect pests. In this study, we isolated a new Bt strain B-JJX,
and demonstrated that its Cry8Ab1 protein, encoded by the cry8Ab1
gene have high toxicity to scarabaeid larvae. This may potentially
provide the new Bt strain for grubs control and add new resource
of cry8 gene for construction of the new engineered microbes or

Y. Zhang et al. / Microbiological Research 168 (2013) 512517

engineered crops that have highly efcient and specic ICP against
grubs.
Acknowledgments
This study was supported by National Natural Science Foundation of China (31272376), Shandong Provincial Natural Science
Foundation (ZR2011CM031), and Tai-Shan Scholar Construction
Foundation of Shandong Province, China. We also would like to
give us great appreciation to Prof. Guoxun Li and Mr. Xinglong Liu
for their making some contribution to the cloning of cry8Ab1 gene.
References
Adams TT, Eiteman MA, Hanel BM. Solid state fermentation of broiler litter for
production of biocontrol agents. Bioresour Technol 2002;82:3341.
Bohlen PJ, Barrett GW. Dispersal of the Japanese beetle (Coleoptera: Scarabaeidae) in
strip-cropped soybean agroecosystems. Environ Entomol 1990;19:95560.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
1976;72:24854.
Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.
Microbiol Mol Biol Rev 1998;62:80713.
Crickmore N. Bacillus thuringiensis toxin nomenclature. http://www.lifesci.
sussex.ac.uk/home/Neil Crickmore/Bt/. [accessed 07.01.13].
Goldberg LJ, Margalit J. A bacterial spore demonstrating rapid larvicidal activity
against Anopheles sergentii, Uranotaenia unguiculata, Culex univeriattus, Aedes
aegypti, and Culex pipens. Mosq News 1977;37:3558.
Koppenhofer AM, Wilson M, Brown I, Kaya HK, Gaugler R. Biological control agents
for white grubs (Coleoptera: Scarabaeidae) in anticipation of the establishment
of the Japanese beetle in California. J Econ Entomol 2000;93:7180.
Krieg A, Huger AM, Langenbruch GA, Schnetter W. Bacillus thuringiensis var. tenebrionis: enneuer, gegenber Larven von Coleopteren wirksamer Pathotyp. Z Angew
Ent 1983;96:5008.
Kuo WS, Chak KF. Identication of novel cry-type genes from Bacillus thuringiensis
strains on the basis of restriction fragment length polymorphism of the PCRamplied DNA. Appl Environ Microbiol 1996;62:136977.
Lereclus D, Arantes O, Chaufaux J, Lecadet M. Transformation and expression
of a cloned -endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett
1989;60:2118.
Liesch PJ, Williamson RC. Evaluation of chemical controls and entomopathogenic
nematodes for control of Phyllophaga white grubs in a Fraser r production eld.
J Econ Entomol 2010;103:197987.
Liu J, Yan G, Shu C, Zhao C, Liu C, Song F, et al. Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against
coleopteran insects. Appl Microbiol Biotechnol 2010;87:2439.
Luo C, Guo XJ, Zang ZL. Species identication of white grubs in lawns around Beijing
and their damage characteristics. Acta Entomol Sinica 2008;51:10812.
Mannion CM, McLane W, Klein MG, Moyseenko J, Oliver JB, Cowan D. Management
of early-instar Japanese beetle (Coleoptera: Scarabaeidae) in eld-grown nursery
crops. J Econ Entomol 2001;94:115161.

517

Michaels TE, Narva KE, Foncerrada L, Bacillus thuringiensis toxins active against
scarab pests. 1996; USP 5554534.
Noguera PA, Ibarra JE. Detection of new cry genes of Bacillus thuringiensis by use of
a novel PCR primer system. Appl Environ Microbiol 2010;76:61505.
Ohba M, Iwahana H, Asnao S, Suzuki N, Sato R, Hori H. A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specic for
scarabaeid beetles. Lett Appl Microbiol 1992;14:547.
Redmond CT, Potter DA. Incidence of turf-damaging white grubs (Coleoptera:
Scarabaeidae) and associated pathogens and parasitoids on Kentucky golf
courses. Environ Entomol 2010;39:183847.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. 2nd ed.
New York: Cold Spring Harbor Laboratory Press; 1989.
Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P. Bacillus thuringiensis: a
century of research, development and commercial applications. Plant Biotechnol
J 2011;9:283300.
Sato R, Takeuchi K, Ogiwara K, Minami M, Kaji Y, Suzuki N, et al. Cloning heterologous expression and localization of a novel crystal protein gene from Bacillus
thuringiensis serovar japonensis strain Buibui toxic to scarabaeid insects. Curr
Microbiol 1994;28:159.
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev
1998;62:775806.
Schnetter W, Mitterller R, Frschle M. Control of the cockchafer Melolontha
melolontha in the Kraichgau with NeemAzal-T/S. In: Keller S, editor. Integrated
control of soil pests. Conference proceeding IOBC/WPRS, vol. 19; 1996. p. 959.
Shu C, Liu R, Wang R, Zhang J, Feng S, Huang D, et al. Improving toxicity of Bacillus thuringiensis strain contains the cry8Ca gene specic to Anomala corpulenta
larvae. Curr Microbiol 2007;55:4926.
Shu C, Yu H, Wang R, Fen S, Su X, Huang D, et al. Characterization of two novel cry8
genes from Bacillus thuringiensis strain BT185. Curr Microbiol 2009a;58:38992.
Shu C, Yan G, Wang R, Zhang J, Feng S, Huang D, et al. Characterization of a novel cry8
gene specic to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela.
Appl Microbiol Biotechnol 2009b;84:7017.
Silva FAB, Costa CMQ, Moura RC, Farias AI. Study of the dung beetle (Coleoptera:
Scarabaeidae) community at two sites: Atlantic forest and clear-cut, Pernambuco, Brazil. Environ Entomol 2010;39:35967.
Suzuki N, Hori H, Ogiwara K, Asano S, Sato R, Ohba M, et al. Insecticidal spectrum of a novel isolate of Bacillus thuringiensis serovar japonensis. Biol Control
1992;2:13842.
Tang QY, Zhang CX. Data Processing System (DPS) software with experimental
design, statistical analysis and data mining developed for use in entomological
research. Insect Sci 2012., http://dx.doi.org/10.1111/j.1744-7917.2012.01519.x.
Wei X, Xu X, Deloach CJ. Biological control of white grubs (Coleopera: Scarabaeidae) by larvae of Promachus yesonicus (Diptera: Asilidae) in China. Biol Control
1995;5:2906.
Xue J, Liang G, Crickmore N, Li H, He K, Song F, et al. Cloning and characterization of a
novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn
borer and other lepidopteran insects. FEMS Microbiol Lett 2008;280:95101.
Xue JL, Cai QX, Zheng DS, Yuan ZM. The synergistic activity between Cry1Aa and
Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa
armigera. Lett Appl Microbiol 2005;40:4605.
Yu H, Zhang J, Huang D, Gao J, Song F. Characterization of Bacillus thuringiensis
strain Bt185 toxic to the asian cockchafer: Holotrichia parallela. Curr Microbiol
2006;53:137.
Zhang H, Yu Z, Deng W. Isolation, distribution and toxicity of Bacillus thuringiensis
from warehouses in China. Crop Protect 2000;19:44954.