Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Available online at www.sciencedirect.com Agricultural Sciences in China 2007, 6(11): 1343-1351

ScienceDirect

November 2007

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China SU Xu-dongl,2,SHU Chang-longl,ZHANG Jiel, HUANG Da-fang3, TAN Jian-xin2and SONG Fu-ping1 I

State Key Laboratory of Biology f o r Plant Diseases and Insect Pestsllnstitute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, P.R.China Agricultural University of Hebei, Baoding 071001, P.R.China Institute of Biotechnology Research, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China

Abstract Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate 22-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Key words: Bacillus thuringiensis, PCR-RFLP, cry gene, Aedes albopictus

INTRODUCTION Bacillus thuringiensis is a gram-positive bacterium, which produces intracellular and distinctively shaped crystal proteins during its spore-forming period. The crystal proteins, also called insecticidal crystal protein (ICPs) (Hofte and Whiteley 1989), have insecticidal activities and can be used to control insect pests. More than 300 crystal protein genes, named cry or cyt, have been cloned, sequenced, and classified as cryl, cry2, ...,cry44, and cytl and cyt2 on the basis of the amino acid sequence homology of their encoded proteins

(Crickmore et al. 1998; Schnepf et aE. 1998). The active spectra of ICPs includes Lepidoptera, Coleopteran, Diptera, Hymenoptera, Homoptera, and Orthroptera insects (Pinto et al. 2003; Schnepf et al. 1998; Tailor et al. 1992) and other invertebrates, such as Acarina, Nematoda, Protozoa, and Platyhelminthes (Hoffmann et al. 1998). However, a significant number of pests are not controlled by these cry proteins, and insect resistance is an increasingly serious problem, particularly for genetically modified plants. Therefore, it is necessary to screen novel toxins to evaluate their effectiveness against insect pests. A large number of B. thuringiensis collections have

Received 15 March, 2007 Accepted 30 July, 2007 Correspondence SONG Fu-ping, Tel: 46-10-62817545,62896634, Fax: +86-10-62894642, E-mail:[email protected]

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been investigated in various countries,including Mexico, the United States, Brazil, Colombia, Egypt, India, Bangladesh, and European countries. In these studies, the distribution of B. thuringiensis strains and cry genes in local wild ecosystems was evaluated, and many strains harboring novel genes were found (Anwar et al. 1997; Arango et al. 2002; Bravo et al. 1998; Ejiofor and Johnson 2002; Iriarte et al. 2000; Merdan and Labib 2003; Prabagaran et al. 2002; Uribe et al. 2003; VilasB6as and Lemos 2004). Wang et al. (2003) summarized the distribution and diversity of cryl, cry2, and cry9 genes in China. Chen et al. (2004) reported that the distribution of cry genes of isolates, including cryl, cry2, cry3, cry4, cry5, cry6, c r y l l , and cry13 genes, from Taiwan of China was dependent on the geographical origin of the sample. However, information regarding the distributionof various strains is still limited and does not cover many distinct geographic regions of China.

MATERIALS AND METHODS Soil sample collection The soil samples were collected from a depth of 5-10 cm to the surface in April 2002, November 2002, and April 2003 during the dry season in the tropical and semitropical regions. The sites were chosen in the south of Hainan Province, China, which has typically tropical climate, and from the middle or north of Yunnan Province, China, which has a typically semitropical climate. The number of soil samples and the collection sites are listed in Table 1. The soil samplesfrom Diaoling Mountain were supplied by Prof. Jiang Chengling from Yunnan University, China.

Isolation of bacterialstrains Ninety-two B. thuringiensis isolates were screened from soil samples by the temperature selection method. Briefly, 1 g soil was added to 100 mL sterile distilled water in a conical flask, and the suspension was shaken for 10 min and continually heated (75°C) for 15 min. Serial dilutions (lO-l, were plated onto 0.5 x LB (Luria-Bertani) agar, and incubated at 30°C for 72 h. A loop of spores and crystals from a single colony with appearance similar to B. thuringiensis was examined with an Olympus BH-2 microscope (Japan). Thirty-three B. thuringiensis isolates were kindly supplied by Northeast Agricultural University, China. They were isolated from Northeast China, which has a temperate and cold climate.

DNA template preparation B. thuringiensis isolates were grown for 12 h on LB agar plates. Cells from a single colony were inoculated with 5 mL LB medium and grown at 30°C for 12 h with vigorous shaking. One milliliter of culture was harvested by centrifugation and washed with ddH,O. The pellet was resuspended in 200 pL ddH,O and incubated in boiling water for 10 min. The cell lysate was briefly centrifuged at 12000 r/min for 1 min and the resulting supernatant was used for PCR amplification.

PCR and RFLP analysis Twenty-six pairs of universal primers were used to detect cryl genes (Kuo and Chak 1996), cryfl genes

Table 1 The number of soil samples and the collection sites Number of

Location of samples

soil samples ~~

Climate

Number of samples containing isolates

Samples with B. thuringiensis isolates (%)

Number of isolates 43

~

334

Jianfeng Mountain, Hainan Province

Tropic

20

6

72

Diaoluo Mountain. Hainan Province

Tropic

5

7

5

30

Luhuitou Mountain, Hainan Province

Tropic

0

0

0

28 64

Hailuo Farm, Hainan Province

Tropic

0

0

0

Shizi Mountain, Yunnan Province

Semitropic

3

5

6

28

Maoniupiug, Yunnan Province

Semitropic

0

0

0

55

Zixi Mountain, Yunnan Province

Semitropic

9

16

23

52

Donghua Farm, Yunnan Province

Semitropic

1

2

3

20

Diaoling Mountain, Yunnan Province

Semitropic

3

I5

12

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Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and

(Song et al. 2003), cry2-cry4 genes, cry10 genes (Song et al. 1998), cry5-cry9 genes, cry11 genes, cryl3-cry14 genes, cryl6-cry19 genes, cry21-cry22 genes, cry24cry30 genes, cry32 genes, cry34-cry35 genes, and cry40 genes (unpublished). PCR was carried out in a 50 pL volume containing 1 pL template DNA, 0.4 mM deoxynucleotide triphosphate, 0.2 pM primer, 1.5 U Tq DNA polymerase and reaction buffer. Amplification was carried out for 32 reaction cycles of denaturing at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 2 min, with an additional step of extension at 72°C for 10 min. A restriction endonuclease reaction was performed in 20 pL volumes with the PCR products (0.5-1.O pg) and 0.5 U restriction endonuclease, with reaction buffer using the protocol described by Sambrook et al. (1989).

SDS-PAGE analysis Cells were grown for 40 h at 30°C in 200 mL 0.5 x LB medium shaking at 230 r/min. The protein composition of parasporal bodies was analyzed by 10% sodium dodecyl sulphate-polyacrylamidegel electrophoresis as described by Sambrook et al. (1989).

Cloning of PCR products PCR products of the isolate harboring novel cry gene were recovered from 0.7% agarose gels with a UNIQ10 spin column DNA gel extraction kit (Shanghai Sangon Biological Engineering Technology ServicesCo., Ltd., China). Purified fragments were ligated with vector-T easy (Shanghai Sangon Biological Engineering Technology Services Co., Ltd).

Electron microscopy Cells were grown on 0.5 x LB agar plates at 30°C for 3 days, and then harvested into sterile distilled water. The suspension was sprayed onto a microscope slide, and then dried. A 2-nm gold coat was added onto these spores and crystal proteins. Samples were examined with a Hitachi S-4100 electron microscope. For transmission electron microscopy, the isolate was grown to the sporangium stage and then fixed in

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glutaraldehyde in phosphate buffer. The suspension was pelleted, dehydrated in an ethanol series, embedded in Duracupan (Sigma, USA), thin sectioned, and stained with lead citrate. The sectioned cells were viewed under a new Bio-TEM H-7500 transmission electron microscopy.

Bioassay Forty-two B. thuringiensis isolates, which had no positive signals with 26 pairs of primers, were tested for toxicity via bioassay. Insecticidal activities against Plutella xylostella (third-instar) were conducted on fresh cabbage leaf disks by leaf dip bioassays (Tabashnik et al. 1993). Insecticidal activities against Chilo supperssalis (fist-instar), Ostrinia nubilalis (firstinstar), Laphygma exigua (first-instar), Heliothis armigera (first-instar), and Mythimna separate (firstinstar) of Lepidoptera, Tribolium castaneum (firstinstar), and Tenibriomolitor (first-instar) of Coleoptera were measured by incorporating a suspension containing two-fold serial dilutions of purified inclusions into the artificial diet. The 22-34 isolate (containing a novel cry30 gene) was tested against Aedes albopictus (second-instar) of Diptera. The bioassays were performed as described by Lee and Gill (1997).

RESULTS Distribution of B. thuringiensis isolates Ninety-two B. thuringiensis isolates were screened from 683 soil samples (Table 1). The percentage of soil samples from Diaoluo Mountain containing B. thuringiensis was 7%, which was higher than in other regions in Hainan Province (Table 1). No isolate was screened from the soil samples in Luhuitou Mountain and Hailuo Farm, Hainan Province, with destroyed or seriously affected vegetation. The proportion of soil from Zixi Mountain containing B. thuringiensis isolates was 16%, which was the highest from any of the regions sampled in Yunnan Province. No B. thuringiensis isolate was screened from the soil samples collected from Maoniuping,Yunnan Province, which has an altitude ranging from 3 300 m

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to 4000 m above sea level. Six B. thuringiensis isolates were obtained from 64 soil samples from Shizi Mountain, Yunnan Province, and 3 B. thuringiensis isolates were screened from 52 soil samples from Donghua Farm, Yunnan Province. Among the 41 total soil samples containing B. thuringiensis, 32 (78%) were collected from primeval forests, 14.6% were obtained from grasslands, and only 7.4% were from agricultural soils. Sunlit sides of mountains contained more strain sources (97.5% of 41 soil samples were collected on the sunlit side).

harbored cry2 genes. The percentage of isolates containing the gene profile of c r y l , cry2 and the gene profile of c r y l , c r y 2 , cry9 were 23 and 2.4%, respectively. The cry8E gene was found in 12 isolates, and the cry30 gene was more abundant, found in 13 isolates. Twelve isolates harbored the gene profile of c r y l l B a and cry30Aa. Forty-two isolates had no positive signal using the 26 pairs of primers for PCR amplification.

The cry gene profiles of B. thuringiensis isolates

Crystal protein composition and crystal morphology

The cry gene profiles of B. thuringiensis isolates were characterized by PCR-RFLP analysis (Table 2). Seventeen cry gene profiles were detailed: of all the isolates, 58 isolates contained cryl genes and 32 isolates

We also assessed the protein contents and shapes of parasporal crystals from 126 isolates. Five protein patterns on the gels were produced by isolates containing cry genes (Table 2). Thirty-two isolates containing both

Table 2 B. thuringiensis isolates' gene combination and analysis of crystal protein') NO.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

cry gene profile

Number of isolates

%*)

Localityl)

crylAa crylAa, crylAc crylAb crylAa, crylAc, crylla crylAa, crylCa crylAb, cry2Ab crylAa, crylAb, cry2Ab crylAa, crylBd, cry2Ab crylAa, c r y l d b , crylAc, cry2Ab crylAa, crylAc, crylla, cry2Ab CryIda, crylAc, crylDb, crylld, cry2Aa, cry2Ab crylAa, c r y l d b , cry2Ab, cry9Ba CrylAb, cry2Ab, cry9Ba cry8Ea c r y l l B b , cry30Aa cry30

5 7 4 8 2 6 4 5 5 8 1 1 2 12 12 1 3 7 2 2 1 1 2 4 4 1 1 5 2 1 1 1 1 2 1

4.0 5.6 3.2 6.3 1.6 4.8 3.2 4.0 4.0 6.3 0.8 0.8 1.6 9.5 9.5 0.8 2.4 5.6 1.6 1.6 0.8 0.8 1.6 3.2 3.2 0.8 0.8 4.0 0.8 0.8 0.8 0.8 0.8 1.6 0.8

N 1 of H, 6 o f N N N N H H H H ?ofH,lofN Y H H Y Y Y N Y Y H H H H H Y F H Y N H N H H H N

Crystal protein ( m a )

130 130 130 130 130, 60 130, 60 130, 60 130, 60 130, 60 130,60 130, 60 130, 60 130, 60 130 60, 50, 30 70, 30 35 140, 60 60, 30 60 60, 20 80, 55 40 130 60 140, 60 55, 30 60 90 35 63 60 130 70, 30 60

Shape of crystal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Spherical Spherical Spherical Spherical Rhomboid Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Bipyramidal Specific Square Square Square Amorphous Amorphous Spherical Spherical Spherical Ovoid Ovoid

I)-, no gene could be amplified with any cry gene primers in isolate. 2)Thestrains containing the same profile of cry genes were observed with different frequency l)N, Northeast China; Y,Yunnan Province; H, Hainan Province.

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Identification and Distribution of Bacillus thurinaiensis Isolates from Primeval Forests in Yunnan and

cry1 and cry2 genes produced 130 and 60 kDa protein bands, and formed bipyramidal crystals. Among 36 isolates with 130 kDa proteins, 24 isolates harboring only cry1 genes formed bipyramidal crystals, and 12 isolates, such as SH49-1, contained the cry8Ea gene and produced spherical crystals (Fig.1, lane 6). The isolate 22-34, containing the novel cry30 genes had a spherical crystal made up of 65 kDa proteins (Fig.2). Nineteen different protein patterns on the gels were found within 42 isolates, but these isolates produced various shapes of crystals that had no positive signals with the primers for PCR test (Table 2). Intriguingly, the strain 51-53, with ovoid crystals, produced 70 and 30 kDa proteins (Fig.1, lane 2, Fig.3-C). The crystals from isolates 569-98, 570-124, D1-63, and 211-1

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connected with spores following cell lysis. Meanwhile, the isolate 569-98 produced a square crystal composed of several proteins of 140 and 60 kDa (Fig.1, lane 3, Fig.3-E). The isolate 570-124 had 35 kDa proteins with an amorphous crystal (Fig.1, lane 5 , Fig.3-F). The isolate D1-63 expressed 130 kDa proteins with small bipyramidal crystals (Fig.1, lane 4, Fig.3-G). However, the isolate Z11-1 had a unique-shaped crystal of 60 kDa proteins enclosed in an envelope (Fig.3-H). All the aforementioned data demonstrated that the size of the crystal protein was determined by cry genes (except silent gene), and different crystal was composed of different protein, the type of cry genes and crystal protein could be speculated according to the shape of the crystal.

Cloned fragment of potentially novel gene One novel cry gene were successfully detected using PCR-RFLP. A fragment (1 419 bp) of a novel cry30 gene in the strain 22-34 was cloned and had DNA sequence homology of 89 and 76% to holotype genes cry30Aa and cry30Ba, respectively. The sequence was blasted at the NCBI website and submitted to the GenBank (the accession number for the gene in this study is AY916046). Fig. 1 SDS-PAGE analysis of crystal proteins in B. thuringiensis isolates. Lane 1, strain 5116-33; lane 2, strain J1-53; lane 3, strain J69-98; lane 4, strain D1-63; lane 5, strain J70-124; lane 6, strain SH49-1; lane 7, strain YD110-1. Molecular masses are given in kDa, Myosin (200 m a ) , P-galactosidase( I 16 m a ) , Phosphorylaseb (97.4 kDa), Serum albumin (66.2 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (31 m a ) .

Toxicity bioassay The strains 22-34 harbored the cry gene which was a novel gene. The Cry30 was usually poisonous to

Fig. 2 The morphasm and protein composition of crystal of strain 22-34. A and B, electron microscopy photos of strain 22-34; s, spore: c, crystal. C, lane 1, the pattern of crystal proteins; M, molecular masses are the same to those in Fig.1.

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SU Xu-dong er al.

Fig. 3 A number of strains had no reaction product with any of the PCR primers. A, strain J116-33 produced a bipyramidal crystal; B, strain YD110-1 had a rhomboid crystal; C, strain 51-53 had an ovoid crystal; D, strain Y45 produced an amorphous;E, strain 569-98 had a square crystal; F, strain J70-124 expressed an amorphous crystal; G, strain D1-63 produced a small bipyramidal crystal; H, 211-1 had a specific shape of crystal enclosed in a envelope. The scale bar is 1 pm.

Diptera. As a result, the strain 22-34 containing a novel cry30 gene, had toxicity towards Aedes albopictus with an LC,, of 7.2317 yg mL-'. Forty-two isolates containing different shaped crystals did not react with any pair of PCR primers, nor did they exhibit toxicity towards Plutella xylostella, Chilo supperssalis, Ostrinia furnacalis, Laphygma exigua, Heliothis armigera, Mythimna separate, Tribolium castaneum, and Tenebriomolitor.

DISCUSSION Of the soil samples collected from Yunnan and Hainan provices, 7.3 and 5.1% contained B. thuringiensis isolates, respectively. B. thuringiensis populations were determinedby environmentalconditions. After analyzing the soil parameters, Anwar et al. (1997) reported that a 1% increase in sand level caused a 0.34% decrease of B. thuringiensis distribution. Likewise, fewer isolates were found in the samples from Hainan Province than from Yunnan Province, most likely because the sandy soil in Hainan Province contains smaller amounts of available water and nutrients. B. thuringiensis were abundantly distributed in the primeval forest, a terrain that possesses a deeper humus layer, particularly in forest areas on the sunlit sides of the mountains (Leckie et al. 2004). In this study, 97.5% of the soil samples

containing B. thuringiensis strains were collected from areas receiving substantial sunlight. However, the percentage of soil samples from Hainan and Yunnan regions containing isolates were much lower than that from other regions in the world. In Columbia and Mexico, 82 and 90% of soil samples contained B. thuringiensis, respectively (Ben-Dov et at. 1999; Uribe et al. 2003). In Bangladesh, 75% of agriculture soil samples contained B. thuringiensis (Anwar et al. 1997). Ejiofor and Johnson (2002) reported that 6% of soil samples collected in South-Central United States contained B. thuringiensis, whereas 13.2% samples from the seven islands of Ryukyusin, Japan contained B. thuringiensis (Ohba et al. 2000). Therefore, the distribution of B. thuringiensis in different regions varies widely. A review of the regional percentages of soil samples with B. thuringienis indicates that the values range from 0 to 97.5% (DeLucca et al. 1981; Martin and Travers 1989). Thus, regional differences in B. thuringiensis distribution should be considered. Yunnan and Hainan provinces and northeast region of China are located in different climate zones, and each provides unique B. thuringiensis resources. The strains containing cry1 genes were widely distributed in regions of the tropical, or temperate or cold climates in China, particularly in the regions covered with primeval forests (Bravo et al. 1998; Uribe et al. 2003). The percentage

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Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and

of cryl genes in Yunnan Province was lower than in Hainan Province. The combinations of cry11 and cryl genes were detected in 17 isolates, which was similar to Wang et al. (2003). The cryl gene has been found frequently in other countries, including Mexico, where 49.5% of isolates contained the cryl gene (Bravo et al. 1998). In the United States, 62% of isolates harbored the cryl genes (Ejiofor and Johnson 2002), and in Colombia, 73% of isolates reacted with the cryl gene primers (Uribe et al. 2003). In the present study, 46% of the total isolates were found to contain cryl genes. By using the PCR-RFLP system to identify cry gene families, we detected cry8 genes in some isolates from soils of broadleaf forest (evergreen), potato field, and the grassland of the semitropics in Yunnan Province. The partial sequence of the wild cry8 gene (1 221 bp) shared 98.8% identity with the cry8Ea holotype gme of B. thuringiensis which was discovered in the temperate climate of North China (unpublished data). The cry8 genes has a wide distribution in China. In Mexico, cry8B and cry8C genes had been detected in the samples collected from different climate zones; the cry8 genes had also been found in Brazil and Colombia, both of which are tropical countries (Uribe et al. 2003; Vilas-BBas and Lemos 2004). These findings indicate that the cry8 genes can be widely distributed in different climate zones in the world. The cry11 and cry30 genes are toxic to Dipterans and were found in soil samples from Yunnan Province. These isolates were screened from the semitropicalrain forests, including valleys, hillsides beside streams, and grasslands. Bravo et al. (1998) also reported that the genes toxic to Dipterans, including cryll genes, were more frequently found in tropical vegetation soil in Mexico. The combination of cry11 genes and cry4 genes in the B. thuringiensis subsp. israelensis had been identified in 1986 (Ibarra and Federici 1986). The cry11 and cry30 genes had also been found in a single strain (Ibarra et al. 2003). Identifying cry genes which were harbored by isolate was only a base on its toxin, because many cry genes may be silent and cannot express crystal proteins. It is a l i t to anticipate the isolate toxicity by identifyingcry genes. We identified a potentially novel gene in one isolate using PCR-RFLP. Various techniques based on PCR

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have been developed and these have become the most powerful approaches to identify the cry gene content to predict the insecticidal activity, and to detect the presence of novel genes (Ceron et al. 1995; JuirezPCrez et al. 1997). Kuo and Chak (1996) and Song et al. (1998,2003) developed a PCR-RFLP method for rapid identification of the cryl, cryll, cry2, cry3, cry4/ cry10 genes, and this technique is used not only to retrieve information regarding the presence of genes in new isolates, but also to discover novel genes. Kuo and Chak (1996) detected six novel cry genes in B. thuringiensis strains using PCR-RFLP and a novel cryll-type gene was found in a standard strain and six isolates using PCR-RFLP to identify cryll-type genes from B. thuringiensis (Song et al. 2003). In this study, the isolate 22-34 was found to contain a novel cry30 gene, sharing 89% sequence identity with the cry30Aa gene. Up to date, more than 200 ICP genes have been cloned and sequenced (Crickmore et al. 1998). The Cryl, Cry2, and Cry9 groups exhibit the strongest activity to Lepidoptera. The CrylB, CrylI, Cry3, Cry7, Cry8 and Cry18 groups are the most toxic to Coleoptera, whereas the Cry4, Cryll, Cryl6, Cryl7, Cryl9, Cry24, Cry25, Cry27, Cry29, Cry30, Cry32, Cry39, Cry40 groups are highly active to Diptera (Frankenhuyzen and Nystrom 2002). But there are also many B. thuringiensis isolates harboring novel cry genes and producing crystals which exhibited no toxicity. Of the 42 isolates that did not react with any PCR primers, many produced distinctive crystals (Fig.3). Four isolates had uniquely shaped crystals, which tightly connected with spores following autolysis (Fig.3-E, -F, -G, -H). These isolates did not exhibit toxicity against the insects tested in this study. Lopez-Meza and Ibarra (1996) identified a B. thuringiensis isolate whose parasporal crystal was enclosed within the spore’s outermost envelope and that also exhibited no toxicity previously. However, the crystal shape of this isolate differed from those of the four isolates in the present study. Vilas-BBas and Lemos (2004) reported that 40.3% of the isolates obtained in Brazil produced no PCR product. Furthermore, in Columbia, the total percentage of the obtained isolates that did not react with any of primer was 17.8% (Uribe et al. 2003). In Mexico, 14%of the obtained isolates yielded no FCR product when assayed with the primers (Bravo et al. 1998). These results

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suggest that throughout the world, there may exist numerous isolates that contain novel and unidentified cry genes. In the present study, the distribution of wild B. thuringiensis resources in Yunnan and Hainan provinces and Northeast China was investigated. Several unique isolates were found to contain novel cry genes and to produce specific proteins. However, we also identified several isolates that did not interact with the 26 pairs of primers used, and were also not toxic to the 8 insect species tested in this study. These findings indicate that there are potentially novel cry genes still to be identified in these isolates, and that their toxicity towards other insect species needs to be evaluated.

Acknowledgements This study was supported by the grants from the National Basic Research Program of China (973 Program) (2003CB 114201), the National High Technology Research and Development Program of China (863 Program,2006AA02Z189,2006AA10A212).

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