Journal of

INVERTEBRATE
PATHOLOGY
Journal of Invertebrate Pathology 82 (2003) 6371
www.elsevier.com/locate/yjipa

Characterization of cry1, cry2, and cry9 genes in

Bacillus thuringiensis isolates from China
Jinhong Wang,a,* Annemie Boets,b Jeroen Van Rie,b and Gaixin Rena
a
Department of Microbiology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, PR China
b
Bayer CropScience N.V., 9000 Ghent, Belgium
Received 21 November 2002; accepted 3 December 2002

Abstract

Bacillus thuringiensis isolates from dierent ecological regions and sources of China were analyzed to study the distribution and
diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and
represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene,
respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%),
cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three dierent cry1 gene proles were de-
tected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1Ccry1D and cry1Acry1I gene
combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on
this RFLP analysis, one novel cry1A-type gene was observed.
2003 Elsevier Science (USA). All rights reserved.

Keywords: Bacillus thuringiensis; Isolates; PCR; cry genes; RFLP

1. Introduction
The growing public concern, stricter environmental
regulations, and buildup of resistant biotypes to syn-
thetic insecticides have led to an increased interest in
alternative environment-friendly insect control strate-
gies. Currently, biocontrol methods have become one of
the most useful tools in integrated pest management. As
commercial biopesticides, products of Bacillus thuringi-
ensis have been successfully used to control various in-
sect pests in the past 50 years (Schnepf et al., 1998). This
organism can synthesize large quantities of insecticidal
crystal proteins (ICPs) during sporulation, and some of
the ICP genes have been applied for bioengineered crop
protection, resulting in transgenic crop plants with ex-
cellent insect protection (Van Rie, 2000).
Bacillus thuringiensis strains are distributed world-
wide in soil, stored products, insects, insect breeding
environments, and the phylloplane. Several B. thuringi-
*
Corresponding author. Fax: +86-22-23508800.
E-mail address: tracywang70@hotmail.com (J. Wang).
ensis ICPs have been found that are highly toxic against
lepidopteran, coleopteran, or dipteran insect species
(Hofte and Whiteley, 1989). Recently, varying levels of
susceptibility of Hymenoptera, Homoptera, Malloph-
aga, Orthoptera, nematodes, mites, and protozoa to
B. thuringiensis strains have been reported (Feitelson,
1993). Up to date, more than 200 ICP genes have been
cloned, sequenced, and classied into 32 groups of cry
genes and 2 groups of cyt genes based on the amino acid
homology of the corresponding proteins (Crickmore
et al., 1998). The Cry1, Cry2, and Cry9 groups exhibit
strongest activity to Lepidoptera; the Cry3, Cry7, and
Cry8 groups are most toxic to Coleoptera; whereas the
Cry4 and Cry11 groups are highly active to Diptera.
Some Cry proteins were reported to display toxicity to
more than one insect order, for example the Cry1I
protein that is toxic to both Lepidoptera and Coleoptera
(Tailor et al., 1992), and the Cry1B protein with activity
against Lepidoptera, Coleoptera, and Diptera (Zhong
et al., 2000). Apart from Cry proteins, some B. thurin-
giensis strains also secrete insecticidal proteins into the
culture medium. These proteins have been referred to as
vegetative insecticidal proteins (Vip proteins) since they

0022-2011/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0022-2011(02)00202-1
64 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371
are expressed during the vegetative stage (Estruch et al.,
1996). However, there are still a signicant number of
important agricultural insect pests that are not sensitive
to the available known B. thuringiensis toxins, and re-
sistance to formulated B. thuringiensis products has been
reported (Tabashnik, 1994). Therefore, the identication
of new B. thuringiensis isolates and the search for novel
genes encoding new insecticidal proteins will remain a
long-term objective for pest control.
Recently, several screening projects of B. thuringiensis
collections from dierent regions have been described
(Ben-Dov et al., 1999, 1997; Bourque et al., 1993; Bravo
et al., 1998; Carozzi et al., 1991; Chak et al., 1994; Ju-
arez-perez et al., 1997; Kim, 2000; Kuo and Chak,
1996). The strategies applied in those programs were
based on the PCR method, which has become one of the
most powerful approaches to identify the cry gene
content, predict the insecticidal activity, and detect the
presence of novel genes.
During the past decades, B. thuringiensis strains have
been isolated from many regions in China (Dai et al.,
1996; Dai et al., 1994; Ren et al., 1975; Wang et al.,
2000). In this paper, we describe 310 B. thuringiensis
strains isolated from soil, stored product dust, dead in-
sects, and leaves from 25 provinces, 2 autonomous re-
gions, and 4 municipalities of China. These strains were
analyzed for the presence of certain ICP genes using a
multiplex PCR method. Following PCR amplication,
restriction fragment length polymorphism (RFLP) was
further applied to identify novel cry1A and cry2 gene
types. Bioassay methods were also utilized to determine
the activity of isolates against Lepidoptera and Cole-
optera in order to guide the search for novel genes en-
coding active proteins.
2. Materials and methods
2.1. Bacillus thuringiensis strains
Strains used as references in the PCR analysis were
provided by Bayer CropScience N.V., Ghent, Belgium.
Natural strains were isolated from soils, stored product
dust, dead insects, and leaves within China by Nankai
University, Tianjin, PR China (Wang et al., 2000).
2.2. PCR analysis
Bacillus thuringiensis strains were grown on Luria
Bertani medium plate for 1618 h. A loop of cells was
transferred to 100 ll ddH2 O and mixed, 5 ll was used as a
DNA template. PCR was carried out in a DNA Thermal
Cycler (MJ Research, Wartertown, MA). The PCR mix-
ture included 250 mM (each) deoxynucleoside triphos-
phate, 50 pM each of the primers, 2.5 U Taq DNA
polymerase enzyme, and 5 ll of 10 PCR buer (Amer-
sham Pharmacia Biotech) in a total volume of 50 ll. Oli-
gonucleotide PCR primers were as described by Wang et
al. (2001) and Wang (2001). The multiplex PCR analysis
and PCR cycling conditions were similar to those de-
scribed by Bravo et al. (1998) and Ben-Dov et al. (1999).
2.3. Identication of cry gene-types by PCRRFLP
The specic primers for detection of a cry gene be-
longing to a particular subfamily were applied in PCR
amplication. The PCR fragment was then digested with
AluI and Sau3AI separately. The digest mixture in-
cluded 5 ll of PCR product, 5 U of the enzyme, and 2 ll
of 10 restriction enzyme buer in a total volume of
20 ll. The digest mixture was incubated at 37 C for 4 h
and digestion products were analyzed by agarose gel
electrophoresis.
2.4. Bioassays
The activities of the spore-crystal mixtures of B.
thuringiensis isolates that do not contain cry1A or cry2
genes were tested on Helicoverpa zea and Heliothis vi-
rescens. Articial diet (Vanderzant, 1962) was dispensed
in wells of Costar 48-well plates. Twenty ve microliters
of sample was applied on the surface of the diet and
dried in a laminar airow. One neonate larva was placed
in each well and 18 larvae were used per sample. Mor-
tality was scored after 7 days.
The activities of the spore-crystal mixtures of all the
310 B. thuringiensis isolates were tested on Diabrotica
virgifera. Articial diet (Sutter et al., 1971) was dis-
pensed in wells of Costar 24-well plates. Fifty microliters
of sample was applied on the surface of the diet and
dried in a laminar airow. Four neonate larvae were
placed in each well, 24 larvae were used per sample.
Mortality was scored after 7 days.
2.5. Denition of the cry gene distribution frequency
The distribution frequency of a cry gene in Bt strains
from a certain origin is dened as the percentage of the
B. thuringiensis isolates containing this gene among all
the isolates from that origin. For example, 141 B. thur-
ingiensis isolates were found in the Temperate zone,
among them 93 strains contained cry1A gene, so the
frequency of cry1A genes of this zone is 65.9% (93/141).
3. Results
3.1. The distribution of B. thuringiensis isolates according
to region and source of isolation
During the past several years, we have collected more
than a 1000 samples from various soils, stored product
 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371
Table 1
Geographical and ecological origin of the samples used to isolate the B. thuringiensis strains analyzed in this study
Location of samples Source of samples
Soil Stored product dust
Temperate zone 16 70
Subtropical zone 56 79
Tropical zone 2 4
Tibet 3 3
Total number 77 156
dust, dead insects, insect feces, and leaves from all the
regions throughout China (Wang et al., 2000). Ap-
proximately 965 B. thuringiensis strains were obtained
from more than 20,000 spore-forming colonies by mi-
croscopic observation.
In this paper, 310 B. thuringiensis strains isolated
from 249 samples (Table 1), which represent isolates
from dierent sources and regions, were characterized
based on an ICP gene analysis. China, with its broad
territory, unique and rich biodiversities, can be divided
into three main geographic and climatic regions (Tem-
perate zone, Subtropical zone, and Tropical zone). The
geographical origin of the B. thuringiensis isolates is
shown in Fig. 1. These selected strains were isolated
from the Temperate zone that occupies about 30% of the
country, the Subtropical zone that occupies more than
60% of the country; and Tropical zone that occupies less
than 10% of the country. For the rst time, data are
reported the analysis of the cry gene content in B.
thuringiensis isolates from Tibet, which belongs to the
high plateau region of the Temperate zone (4600 m
above the sea level). Since the geographic features of this
province are unique, the isolates from it were treated as
Fig. 1. Geographical origin of the B. thuringiensis isolates from China.
The rhombus symbols represent the distribution of the B. thuringiensis
isolates that were used in this paper for extensive gene analysis.
65
Total number
Dead insects Leaves
7 3 96
2 3 140
1 0 7
0 0 6
10 6 249
a separate group to compare with those from the other
three main zones.
The overview of the geographical origin of the 310
isolates from dierent sources is shown in Table 2. Most
isolates originate from the Temperate zone (141 isolates)
and Subtropical zone (151 isolates), while much fewer
isolates were derived from samples from the Tropical
zone and from Tibet (nine isolates in both cases). With
respect to the biological origin of the isolates, more
isolates were derived from soil and stored product dust
samples than from insect and leaf samples. In view of
the small sample size for the isolates from two geo-
graphical origins (Tibet and the Tropical zone) and from
2 biological origins (insects and leaves), the results with
respect to the cry gene distribution in these isolates may
not necessarily be representative of this region or source.
3.2. Identication of cry1, cry2, and cry9 gene composi-
tion of B. thuringiensis isolates
The cry gene composition of the B. thuringiensis
isolates was determined by PCR using general primers
for cry1, cry2, and cry9 genes. The cry1 gene positive
isolates were further analyzed with specic primers for
cry1A, cry1B, cry1C, cry1D, cry1E, cry1F, cry1G,
cry1H, cry1I, cry1J, and cry1K genes by multiplex PCR.
Strains containing cry1-type genes were the most
abundant and represent 237 of the 310 B. thuringiensis
isolates (76.5%) (Fig. 2). About 70% of the isolates con-
tained a cry2 gene and 15.5% contained a cry9 gene.
Among the cry1 gene containing isolates, 90.7% strains
also harbored a cry2 gene, while 99.1% of the cry2 gene
containing isolates also harbored a cry1 gene. Further-
more, it is interesting to nd that among the 48 cry9 gene
containing isolates, 87.5% did not harbor a cry1-type gene
and 83.3% did not harbor either a cry1-type gene or a
cry2-type gene. These results show that while the cry1 and
cry2 genes have a high tendency to occur together, there is
no such association between cry9 genes and cry1 or cry2
genes. Ten percent of the strains did not contain a cry1,
cry2, or cry9 gene. All the dierent cry1 subfamilies were
observed with dierent frequencies. Among them, cry1A
(67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%)
genes were the most abundant, while cry1B (12.9%),
66 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371

Table 2
Geographical and ecological origin of B. thuringiensis isolates
Geographical origin of isolates Ecological origin of isolates Total number
Soil Stored product dust Dead insects Leaves
Temperate zone 22 106 10 3 141
Subtropical zone 65 80 3 3 151
Tropical zone 2 7 0 0 9
Tibet 4 5 0 0 9
Total number 93 198 13 6 310

Table 3
cry1 gene proles of B. thuringiensis in China
No. of cry gene proles No. of Frequency
proles isolates (%)
1. 1A + 1C + 1D + 1I 82 34.6
2. 1A + 1I 18 7.59
3. 1A + 1C + 1I 15 6.33
4. 1A + 1C 13 5.49
5. 1A + 1C + 1D 12 5.06
6. 1A 11 4.64
7. 1A + 1D + 1I 10 4.22
8. 1A + 1E + 1I 10 4.22
9. 1A + 1B + 1E + 1G + 1I 6 2.53
10. 1A + 1B+1I 6 2.53
11. 1I 5 2.11
12. 1A + 1B + 1G + 1I 4 1.69
Fig. 2. The cry gene composition of B. thuringiensis isolates. 13. 1C + 1D + 1I 4 1.69
14. 1D + 1I + 1J + 1K 4 1.69
15. 1A + 1B + 1C + 1G + 1I 3 1.27
cry1E (8.1%), cry1G (7.7%), cry1J (4.8%), and cry1K 16. 1A + 1B + 1C + 1D + 1I 2 0.84
17. 1A + 1B + 1D + 1H + 2 0.84
(3.2%) genes were less prevalent. Very few cry1H (0.9%)
1I + 1J
and cry1F (0.3%) genes were found. 18. 1A + 1C + 1K 2 0.84
19. 1A + 1D 2 0.84
3.3. Diversity of cry1 gene combinations of B. thuringi- 20. 1E 2 0.84
ensis isolates 21. 1E + 1I 2 0.84
22. 1A + 1B + 1C + 1E + 1 0.42
1G + 1I
Forty-three dierent cry1 gene proles were detected in 23. 1A + 1B + 1C + 1G + 1 0.42
237 cry1 containing B. thuringiensis isolates (Table 3). 1I + 1J
This indicates a high diversity in the cry gene content of B. 24. 1A + 1B + 1D + 1E + 1 0.42
thuringiensis strains from China. More than 90% of the 1G + 1I
25. 1A + 1B + 1D + 1H + 1I 1 0.42
cry1 isolates harbored more than one type of cry1 gene.
26. 1A + 1B + 1D + 1I 1 0.42
The most common prole was the cry1A, cry1C, cry1D, 27. 1A + 1B + 1E 1 0.42
and cry1I gene combination that represented 82 of the 237 28. 1A + 1B + 1E + 1G 1 0.42
(34.6%) cry1 isolates. Strains containing a cry1A and a 29. 1A + 1B + 1E + 1I 1 0.42
cry1I gene (7.6%) or a cry1A, cry1C, and cry1I gene 30. 1A + 1B + 1G 1 0.42
31. 1A + 1B + 1G + 1I + 1J 1 0.42
(6.3%) were also frequently observed. We found several
32. 1A + 1B + 1G + 1J + 1K 1 0.42
cry1 genes that were associated at a high frequency. For 33. 1A + 1G + 1I 1 0.42
instance, 82% of the cry1C gene containing isolates also 34. 1B + 1G + 1I 1 0.42
contained a cry1D gene, and a cry1I gene was present in 35. 1B + 1G + 1I + 1J 1 0.42
79% of the cry1A containing isolates. 36. 1B + 1G + 1I + 1J + 1K 1 0.42
37. 1B + 1I 1 0.42
38. 1B + 1I + 1J 1 0.42
3.4. Distribution of cry genes in dierent ecological 39. 1B + 1J 1 0.42
regions 40. 1C 1 0.42
41. 1D + 1I + 1K 1 0.42
The distribution of cry genes in dierent ecological 42. 1F + 1I + 1J + 1K 1 0.42
43. 1J 1 0.42
regions of China was analyzed (Fig. 3). All the observed
 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371 67

Fig. 4. The cry gene distribution according to sample origin.

Fig. 3. The cry gene distribution in dierent ecological regions.

cry genes can be divided into three groups. The rst
group consists of cry2, cry1A, cry1I, cry1C, and cry1D
genes, which were the ve most common genes found in
all the three main zones as well as in Tibet. Among them,
the cry2, cry1A, and cry1I genes were present at almost
the same frequencies in the Temperate zone and Sub-
tropical zone, whereas more isolates containing cry1C
and cry1D genes were observed in the Temperate zone.
The second group includes cry1B, cry1E, cry1G, cry1J,
cry1K, and cry9 genes, which were less abundant and
distributed dierently in each region. For example, the
cry1B, cry1E, and cry1G genes were present more fre-
quently in the Subtropical zone than in the Temperate
zone, while the cry1J and cry1K genes were present at
similar frequency in these two zones. We also found that
the frequency of cry1B and cry1J was signicantly
higher in the Tropical zone (44.4 and 33.3%, respec-
tively) than in the Temperate (6.4 and 4.3%, respectively)
and Subtropical zone (17.9 and 4.0%, respectively).
However, more isolates from the Tropical zone should

Table 4
PCRRFLP digest patterns of known cry1A family genes
cry gene Size of PCR fragment (bp) Digest patterna
AluI Sau3AIa
1Aa 1458 2466158184288346354 1759120138160926
1Ab 1500 184224315346354 1759120138160929
1Ac 1504 6224315321560 2971129
1Ad 1420 24158346354538 82155197297689
1Ae 1423 224315346538 17120160197362567
a
The numbers in the digest pattern column represent the size (in bp) of the restriction fragments.

Fig. 5. (A) and (B) are the restriction patterns by AluI and Sau3AI, respectively. Lane 1: molecular size marker (k DNA PstI digest); lane 2: 100-bp
DNA ladder; lanes 39: digested cry1A amplicons from dierent B. thuringiensis isolates; lanes 1012: digested amplicons from cry1Aa, cry1Ab, and
cry1Ac gene Escherichia coli clones. Lane 8: the digested amplicon of the B. thuringiensis isolate with a novel pattern: the Sau3AI digest pattern is the
same as the cry1Ac gene, but the AluI digest pattern is clearly dierent.
68 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371

be studied in order to assess whether this higher fre-

+
)
)
)
9
quency is a true characteristic of this zone. The last
group contains the two infrequent genes, the cry1H and

1K
cry1F genes. There was only one isolate from the Tem-

+
+

+
)
perate zone containing a cry1F gene; the 3 cry1H con-
taining isolates were found in the Subtropical and

1J
+
+
+
+
Tropical zone. Finally, it is interesting to note that the
isolates, which did not contain any of the studied gene

1I
+
+
+
+
families (cry1, cry2, and cry9 genes), were observed at a
similar frequency (around 10% of the strains) in each

1H
region of China.

)
)
)
)
3.5. Distribution of cry genes in dierent sources

1G
)
)
)
)
The analysis of cry gene distribution according to the

1F
+
)
)
)
sample source of the B. thuringiensis isolates is shown in
Fig. 4. The frequencies of studied genes were compared

1E
only for the soil and stored product dust isolates. We

)
)
)
)
found that the isolates containing the common genes
(cry2, cry1A, cry1I, cry1C, and cry1D genes) and most

1D

+
)

)
of the less abundant genes (cry1B, cry1E, cry1G, cry1J,

The cry gene content of isolates

and cry1K genes) were more frequently derived from the

1C
)
)
)
)
stored product dust samples than that from the soil
samples. In addition, the two infrequent genes (cry1H

1B
and cry1F) were both present in the stored product dust

+
)
)

)
isolates, but absent from soil isolates. Only the isolates
containing a cry9 gene and those containing none of the

)
)
)
)
2
studied genes were present at a higher frequency in the
soil samples.
1A
)
)
)
)
The sign / indicates that the bioassay has been repeated with the same preparation.
3.6. Detection of novel genes encoding Lepidoptera-active
ICPs Subtropical
Temperate
Temperate
Tropical

Two methods were applied in order to detect novel

Zone

genes encoding Lepidoptera-active ICPs: PCRRFLP

analysis on selected cry genes and bioassays combined
with more general cry PCR analysis. PCRRFLP
analysis was rst employed to identify the type of cry1A
Source
Origin
Some characteristics of the four active B. thuringiensis isolates

Dust

Dust
Dust
Dust

or cry2 gene in all of the 243 isolates containing one or

both of these two genes. The specic primers for the
cry1A gene family were applied in PCR amplication,
and the resulting amplicons (1.41.5 kb) were then di-
Activity of spore-crystal

gested with AluI and Sau3AI separately. The expected

mixture (% mortality)
H.z.b

6 grd
11gr

length of the PCR product and RFLP digest pattern of

22
6

the known cry1A genes is shown in Table 4. One isolate

with an apparently novel restriction pattern was ob-
served: the Sau3AI digest pattern of this isolate (Fig. 5B,
gic

gr
gi
gi

lane 8) is same as the cry1Ac gene (Fig. 5B, lane 12);

72/31

77/61
83/77

71/41
H.v.a

while its AluI digest pattern consists of a 158-, 288-, 315-

Growth retardation.
Growth inhibition.

, and 560-bp fragment (Fig. 5A, lane 8), is dierent from

the cry1Ac gene (Fig. 5A, lane 12) and other known
H. virescens.
No. of isolates

cry1A genes, which indicating that it contains a novel

cry1A gene. A similar analysis on the cry2 genes failed to
H. zea.
NK317
NK318
NK467
NK482

uncover any novel gene belonging to this family.

Table 5

Most of the proteins encoded by cry1A-type and

b

d
a

cry2-type genes are known to be active on heliothine

 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371
pests. So only spore-crystal mixtures from isolates that
were negative for both genes were tested on two Lepi-
doptera targets (H. virescens and H. zea). Four isolates
(NK317, NK318, NK467, and NK482) exhibited tox-
icity against H. virescens. Some of the characteristics of
these strains are shown in Table 5.
The data revealed that each of these isolates con-
tained several cry genes other than cry1A or cry2 that
encode ICPs belonging to Lepidoptera-specic Cry
protein families. The absence of cry1A and cry2 genes
was conrmed by PCR. Some or all of these ICPs
might be responsible for the observed activity against
H. virescens, since all of them contain a cry1I and
cry1J gene, so it would be useful to test Cry1I and
Cry1J toxins. The four strains were all isolated from
stored product dust. NK317 and NK318 were isolated
from the Temperate zone, whereas NK467 and NK482
were isolated from the Tropical and Subtropical zone,
respectively. No isolate with signicant activity against
H. zea was found.
3.7. Detection of novel genes encoding Coleoptera-active
ICPs
As for the lepidopteran pests, bioassays followed by
PCR analysis can potentially indicate the presence of
novel genes active on Coleoptera. Therefore, the spore-
crystal mixtures of all 310 B. thuringiensis isolates were
tested on D. virgifera, which is one of the most eco-
nomically important coleopteran pests. None of spore-
crystal mixtures active on this target was found.
4. Discussion
In this paper, the presence of certain cry genes was
analyzed in 310 B. thuringiensis isolates from dierent
regions and various sources of China. These results are
useful for understanding the distribution of cry genes
and may lead to the identication of novel candidate
genes for bioengineered crop protection (Van Rie,
2000).
The cry gene content of the B. thuringiensis isolates
from China exhibited a wide diversity. According to the
PCR analysis, all the cry1 gene subfamilies, as well as
cry2 and cry9 genes were observed in our collection.
Among them, the cry1-type genes were the most abun-
dant in every region and source. This trend has also been
found in other studies (Ben-Dov et al., 1997; Bravo
et al., 1998; Kim, 2000). The cry2 genes represented the
second most frequent gene family, whereas cry9 genes
displayed the lowest frequency. The analysis of these
three cry gene families showed that the cry1 and cry2
genes were most often present together, which is similar
to the results of other reports (Ben-Dov et al., 1997;
Kim, 2000; Zhang et al., 2000). In our study, we ob-
69
served a higher frequency of cry9 genes (15.5%) than
that reported from other collections. For instance, in a
496 B. thuringiensis collection from Mexican soil sam-
ples, Bravo et al. (1998) detected cry9 genes in 2.6% of
the strains. Ben-Dov et al. (1997) found in their 215 Bt
collection from soil samples from Israel, Kazakhstan,
and Uzbekistan, that 10.2% of the strains harbored cry9
genes. In addition, whereas the cry9 gene containing
isolates in our collection seldom contained a cry1 or cry2
gene, Ben-Dov et al. (1999) found that all their cry9 gene
containing strains also harbored a cry1 and a cry2 gene.
Within the cry1 gene family, the cry1A, cry1I, cry1C,
and cry1D genes were most frequent, cry1B, cry1E,
cry1G, cry1J, and cry1K were less frequent and very few
cry1H and cry1F genes were observed in our collection.
This result deviates from other studies. For example,
Bravo et al. (1998) found that the cry1Ba gene was
frequently observed, while very few cry1E genes were
discovered within the cry1 family. The cry genes iden-
tied in Korea by Kim (2000) revealed that cry1A,
cry1C, and cry1D genes were the most predominant in
their collection, while cry1B, cry1E, cry1F, and cry1G
genes were less frequent, and no cry1I gene was found.
Furthermore, the analysis of 225 B. thuringiensis soil
isolates from Taiwan by Chak et al. (1994) showed that
cry1A, cry1C, and cry1D genes were present most fre-
quently while no strains containing cry1B, cry1E, and
cry1F genes were found.
The reported cry gene distribution among studies and
collections is quite variable. There are some cry genes
that have been identied commonly everywhere in the
world, such as cry1 type genes. On the other hand, some
cry genes are presented more frequent in one region than
in another. For example the cry1A, cry1C, cry1D, and
cry2 genes were found more commonly in isolates from
Asia (Ben-Dov et al., 1997; Chak et al., 1994; Kim, 2000;
Zhang et al., 2000) than those from Latin America
(Bravo et al., 1998). Furthermore, even collections of Bt
strains isolated from the same country may vary in the
frequency of observed cry genes. For instance, we found
that more isolates harbored cry1C (48.9%), cry1D
(45.5%), and cry1B (16.7%) genes and much less strains
contained a cry1E gene (11.6%) than the results de-
scribed by Zhang et al. (2000). Such variation of cry
gene distribution between studies is likely associated
with dierences in the biological, geographical and
ecological features of the sample.
In this paper, we found that most of the B. thuringi-
ensis isolates contained more than one type of cry gene.
Forty-three distinct cry1 gene proles were identied in
our collection, which indicates the high diversity in the
cry gene contents of the B. thuringiensis isolates. We also
noticed that several cry genes were strongly associated,
such as cry1D/cry1C and cry1A/cry1I genes. The bio-
logical signicance of such associations, if any, is not
clear.
70 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371
Based on PCR screening results of the isolates from
the soil and stored product dust, all the studied genes
were discovered in isolates from dust samples, while no
cry1F and cry1H genes were present in the isolates from
soil samples. In addition, much higher frequencies of all
the cry1 subfamily genes were observed in strains from
dust samples than that in those from soil samples (Fig.
4). The four cry1A and cry2 negative isolates, that are
toxic against H. virescens, were also isolated from the
stored product dust. These data may indicate that cry
gene diversity is somewhat higher in stored product dust
than in soil samples. More extensive studies would be
necessary to conrm this suggestion. Furthermore, in
our previous study (Wang, 2001; Wang et al., 2000), we
found that the average frequency of B. thuringiensis
isolates derived from dust samples (47.7%) was higher
than that from soil samples (29.8%). This observation
implies that the stored product dust is a richer source of
B. thuringiensis strains than soil and conrms previous
ndings (Meadows, 1993).
Among the thirty-one B. thuringiensis isolates that
did not harbor any of the cry1, cry2, or cry9 genes, most
were found in soil samples. This observation was con-
sistent across the four regions of China. Since all of
them did produce crystals, this implied that they might
contain known genes belonging to other families or
potentially novel ICP genes.
Cry proteins are very selectively active against certain
insect species, so the strains containing several types of
cry genes encoding highly active ICPs (Table 3) might be
predicted to have a wider pest spectrum or increased
activity. This would lead to additional broad spectrum
and highly toxic B. thuringiensis insecticides. The char-
acterization of the observed novel cry1A gene and the
search for additional novel genes will be continued.
References
Ben-Dov, E., Wang, Q., Zaritsky, A., Manasherob, R., Barak, Z.,
Schneider, B., Khamraev, A., Baizhanov, M., Glupov, V., Marga-
lith, Y., 1999. Multiplex PCR screening to detect cry9 genes
in Bacillus thuringiensis strains. Appl. Environ. Microbiol. 65,
37143716.
Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R., Manashe-
rob, R., Khamraev, A., Troitskaya, E., Dubitsky, A., Berezina, N.,
Margalith, Y., 1997. Extended screening by PCR for seven cry-
group genes from eld-collected strains of Bacillus thuringiensis.
Appl. Environ. Microbiol. 63, 48834890.
Bourque, S.N., Valero, J.R., Mercier, J., Lavoie, M.C., Levesque,
R.C., 1993. Multiplex polymerase chain reaction for detection and
dierentiation of the microbial insecticide Bacillus thuringiensis.
Appl. Environ. Microbiol. 59, 523527.
Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A.,
Ortiz, M., Lina, L., Villalobos, F.J., Pena, G., Nunez-Valdez,
M.E., Soberon, M., Quintero, R., 1998. Characterization of cry
genes in a Mexican Bacillus thuringiensis strain collection. Appl.
Environ. Microbiol. 64, 49654972.
Carozzi, N.B., Kramer, V.C., Warren, G.W., Evola, S., Koziel, M.G.,
1991. Prediction of insecticidal activity of Bacillus thuringiensis
strains by polymerase chain reaction product proles. Appl.
Environ. Microbiol. 57, 30573061.
Chak, K.F., Chao, D.C., Tseng, M.Y., Kao, S.S., Tuan, S.J., Feng,
T.Y., 1994. Determination and distribution of cry-type genes of
Bacillus thuringiensis isolates from Taiwan. Appl. Environ. Micro-
biol. 60, 24152420.
Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J.,
Lereclus, D., Baum, J., Dean, D.H., 1998. Bacillus thuringiensis
toxin nomenclature. http://www.biols.susx.ac.uk/Home/
Neil_Crickmore/Bt/index.html.
Dai, S.Y., Gao, M.Y., Li, X.G., Li, R.S., 1996. Distribution of Bacillus
thuringiensis in soils of north and south of China. Acta Microbi-
ologica Sinica 36, 295302.
Dai, L.Y., Wang, X.P., Yang, G.Y., Zhang, W.R., 1994. Studies on the
ecological distribution of Bacillus thuringiensis in forest soils of
China. Acta Microbiologica Sinica 34, 449456.
Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A.,
Kozoel, M.G., 1996. Vip3A, a novel Bacillus thuringiensis vegeta-
tive insecticidal protein with a wide spectrum of activities against
lepidopteran insects. Proc. Natl. Acad. Sci. USA 93, 53895394.
Feitelson, J.S., 1993. The Bacillus thuringiensis family tree. In: Kim, L.
(Ed.), Advanced Engineered Pesticides. Marcel Dekker, New York,
NY, pp. 6372.
Hofte, H., Whiteley, H.R., 1989. Insecticidal crystal proteins of
Bacillus thuringiensis. Microbiol. Rev. 53, 242255.
Juarez-perez, V.M., Ferrandis, M.D., Frutos, R., 1997. PCR-based
approach for detection of novel Bacillus thuringiensis cry genes.
Appl. Environ. Microbiol. 63, 29973002.
Kim, H.S., 2000. Comparative study of the frequency, agellar serotype,
crystal shape, toxicity, and cry gene contents of Bacillus thuringiensis
from three environments. Curr. Microbiol. 41, 250256.
Kuo, W.S., Chak, K.F., 1996. Identication of novel cry-type genes
from Bacillus thuringiensis strains on the basis of restriction
fragment length polymorphism of the PCR-amplied DNA. Appl.
Environ. Microbiol. 62, 13691377.
Meadows, M.P., 1993. Bacillus thuringiensis in the environment:
ecology and risk assessment. In: Entwistle, P.F., Cory, J.S., Bailey,
M.J., Higgs, S. (Eds.), Bacillus Thuringiensis, An Environmental
Biopesticide: Theory and Practice. John Wiley, Chichester, En-
gland, pp. 193219.
Ren, G.X., Li, K.T., Yang, M.H., Yi, X.M., 1975. The classication of
the strains of Bacillus thuringiensis group. Acta Microbiologica
Sinica 15, 292301.
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis
and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62,
775806.
Sutter, G.R., Krysan, J.L., Guss, P.L., 1971. Rearing the Southern
corn rootworm on articial diet. J. Econ. Entomol. 64, 6567.
Tabashnik, B.E., 1994. Evolution of resistance to Bacillus thuringiensis.
Annu. Rev. Entomol. 39, 4779.
Tailor, R., Tippett, J., Gibb, G., Pells, S., Pike, D., Jordan, L., Ely, S.,
1992. Identication and characterization of a novel Bacillus
thuringiensis delta-endotoxin entomocidal to coleopteran and
lepidopteran larvae. Mol. Microbiol. 6, 12111217.
Van Rie, J., 2000. Bacillus thuringiensis and its use in transgenic insect
control technologies. Int. J. Med. Microbiol. 290, 463469.
Wang, J.H., 2001, Biodiversity of Bacillus thuringiensis from China
and novel cry gene cloning and expression. PhD thesis, Nankai
University, PR China.
Wang, J.H., Boets, A., Van Rie, J., Ren, G.X., 2001. Characterization
of cry gene distribution and detection of novel genes in Bacillus
thuringiensis isolates from China. In: Program and Abstracts of 4th
Pacic Rim Conference on the Biotechnology of Bacillus thurin-
giensis and its Environmental Impact. Canberra, Australia, p. 61.
 J. Wang et al. / Journal of Invertebrate Pathology 82 (2003) 6371 71

Wang, J.H., Wu, W.H., Chen, Y.H., Ren, G.X., 2000. The ecology
distribution of Bacillus thuringiensis and cry gene diversity in
China. In: Abstracts of 5th International Conference on Bacillus
thuringiensis. Society for Invertebrate Pathology, Guanajuato,
Mexico, p. 98.
Zhang, H.Y., Yu, Z.N., Deng, W.X., 2000. Composition and
ecological distribution of Cry proteins and their genotypes of
Bacillus thuringiensis isolates from warehouses in China. J. Inver-
tebr. Pathol. 76, 191197 (doi:10.1006/jipa.2000.4970).
Zhong, C., Ellar, D.J., Bishop, A., Johnson, C., Lin, S., Hart, E.R., 2000.
Characterization of a Bacillus thuringiensis delta-endotoxin which is
toxic to insects in three orders. J. Invert. Path. 76, 131139.
Vanderzant, E.S., 1962. Rearing the bollworm on articial diet. J.
Econ. Entomol. 55, 140.