Comparison Study Of Toxicity Bacillus thuringensis from Soil Isolate and Mulberry Leaves in Indonesia

Background and Objective: Most strains of B. thuringiensis produce delta-endotoxin crystals toxic to lepidopteran insects such moth. But some strains of B. thuringiensis produce delta-endotoxin crystals toxic to dipteran insects such as mosquitoes and blackflies. To compare B. thuringiensis isolate from soil and mulberry leaves. Material and Methods:One gram of soil samples was suspended in 9 ml of sterile distilled water and shaken for 5 min. the upper layer of the soil suspension was transferred to a test tube and heated at 80°C for 5 min in water bath to kill non-spore-forming organism and vegetative cells to prepare the sporulated culture, bacteria were grown on nutrient agar pH 7.2, at 30°C for 4 days. The isolates were obtained from mulberry leaves collected in West Java, Indonesia, using the leaf-lift technique. Leaves were trimmed to fit inside a 100 mm petri dish. Abaxial leaf surfaces were placed in contact with nutrient agar, and a sterile, per forated stainless steel disk was placed on the leaf sections to ensure maximum contact with the agar. After the sample was coated with carbon and gold, it was observed and photographed with (SEM). However they had differences form crystal protein, but they had unique as insecticidal to control the same orders (mosquitocidal). Results:The study found that B. thuringiensis serovar entomocidus INA288 produced large cuboidal-form crystals and B. thuringiensis serovar aizawai BUN 1-14 a little homology with serovar entomocidus INA288 but, B. thuringiensis israelensis ONR60A has irregular shaped crystal protein. Toxicity results that serovar entomocidus INA288, serovar aizawai BUN 1-14 and serovar israelensis ONR60A showed toxic to mosquito insects. which was composed of major protein of 130 kDa peptides. B. thuringiensis serovar entomocidus INA288 has 70 kDa and aizawai BUN 1-14 had 69 kDa. Conclusion: B. thuringiensis strain had different crystal protein form but had same toxicity.


Introduction
Numerous chemical insecticides have been used to control some insects. While chemical insecticides have knock down effect, they are too expensive harmfull to both humans and the environment. In addition, target insect pests develop biological resistance rapidly especially at higher rates of application. Thus, the increase in pesticidal application to control this pest has urged to researcher to search for biological control alternatives that would be a good component of Integrated Pest Management [1,32,33].
Bacillus thuringiensis is a gram-positive, spore-forming bacterium that produce parasporal crystal during the sporulation stage. The crystal is made of one or more proteins toxic to some insect species. Most strains of B. thuringiensis produce delta-endotoxin crystal toxic to lepidopteran insect such as moth [2,28,29].
The name proposed of Bacillus thuringiensis for a species of bacillus which was isolated from diseased larvae of Mediterranean flour moth Angasta (Ephestia) kuhniella Zell [3]. Later, noted infection of the larvae after the ingestion of the bacillus or its spores, described and named it Bacillus thuringiensis [3,4]. It isolated the same bacillus from the same insect host [4], which had found earlier. This strain is now maintained as B. thuringiensis serovar thuringiensis (serotype H-1). They noticed that the vegetative remains of sporulating cells assumed a rhomboid shape [3,4]. He described this crystalline inclusion in the sporangium of the organism and made further interpretations of the data being accumulated on this bacillus at that time. Neither Berliner nor Mattes attributed those parasporal bodies any role in the the disease process caused by the ingestion of sporalating B. thuringiensis [5].

Materials and Methods
Bacterial stains. The strain of B. thuringiensis used in the present study were B. thuringiensis serovar entomocidus INA 288, serovar aizawai BUN 1-14 and israelenis ONR60A (standard isolate).

Isolation and Identification
B. thuringiensis serovar entomocidus INA288 which had been isolated from Indonesia soil, was prepared according to the method [14]. One gram of soil samples was suspended in 9 ml of sterile distilled water and shaken for 5 min. the upper layer of the soil suspension was transferred to a test tube and heated at 80°C for 5 min in water bath to kill non-sporeforming organism and vegetative cells to prepare the sporulated culture, bacteria were grown on nutrient agar pH 7.2, at 30°C for 4 days. Formation of spores and parasporal inclusion were monitored with a phase-contrast microscope. The culture was scratched on the agar slant as a stock. B. thuringiensis serovar aizawai BUN 1-14 which had been isolated from mulberry leaves. Isolates were obtained from mulberry leaves collected in West Java, Indonesia, using the leaf-lift technique [15,16]. Leaves were trimmed to fit inside a 100 mm petri dish. Abaxial leaf surfaces were placed in contact with nutrient agar, and a sterile, per forated stainless steel disk was placed on the leaf sections to ensure maximum contact with the agar. The lid was replaced, and samples were incubated at 30°C overnight. To prepare the sporulated culture, bacteria were grown on nutrient agar, pH 7,0, at 30°C for 4 days. Formation of spores and parasporal inclusion were monitored with a phase-contrast microscope.

Morphology of Parasporal Inclusion
Isolates were examine with a HITACHI S-800 Scanning Electron Microscope (SEM) at a magnification of 10,000x, according to the method presented by [23,24]. B. turingiensis serovar entomocidus INA288 were cultured on N-broth agar at 30 0 C until almost all cells lysed (overnight). The crystal and spores (about 100 mg wet weight) were washed in 10 ml of 50 mMTris-HCL (pH 8.0). The final precipitate was resuspended in 1 ml of distilled water, and 20 ul of the suspension was air-dried on a glass disk (O 10 mm). After the sample was coated with carbon and gold, it was observed and photographed with SEM.

Biological Activity
The strain were examined for their larvicidal against the larvae of the silkworm, Plutella xylostella and Spodoptera litura. The insect cultures were maintained in this laboratory. Toxicity test with the Leppidopteran insect, B. mori, P. xyloetella and S. litura, were done by introducing ten 3 rdintar larvae were fed on an artificial diet dropped with 0.3 ml of the bacterial suspension and rear at 25°C for 48 hr to determine mortality. The B. thuringiensis isolates were examined for oral insecticidal activity against the insects were prepared by the following procedures. Overnight culture of serovar entomocidus INA288, BUN 1-14 and israelensis ONR60A were grown on 2 ml of nutrient broth at 30°C using tube glass. Then, 200 ul of the overnight culture was plating on nutrient agar, reincubated for 4 days at 30°C. Sporulated cultures were harvested by centrifugation at 10,000 g for min at 4°C. The pellet was washed three times by centrifugation in mMTris-HCL and 1 M NaCL at 4°C, the bacterial suspensions were finally suspended in 500 ul of sterile distilled water. The bacteria were also tested against larvae of the mosquitoes, Aedes aegypti, Aedes japonicus and Culex quinquefasciatus. Ten 2 nd -instar larvae were placed in a test tube containing 10 ml of the spore-parasporal inclusion suspension, respectively, under levels 1 ul/ml. The tubes were kept at 22°C for 24 hr without feeding.

Result and Discussion
In order to identify serovar entomocidus INA288 strains by H-serotype cell with broth, they were dropped to glass slide and the motility of cells was observed under phase-contrast microscope. Since isolate of serovar entomocidus INA288 gave positive reaction in the H 6 serum agglutination test, it was identified as B. thuringeisis serovar entomocidus INA288. interestingly, entomocidus INA288 produced large cuboidal-form crystals (Figure 1). On the other hand, B. thuringiensis serovar aizawai BUN 1-14 a little homology with serovar entomocidus INA288 (Figure 2). Interestingly, B. thuringiensis serovar israelensis ONR60A [23,24] has irregular shaped crystal protein. However, [27] demonstrated that using antibodies of B. thuringiensis serovar israelensis ONR60A showed weakly cross-reactivity to serovar jegathesan. Indeed, B. thuringiensis serovar jegathesan included cryIVD and showed immunological similarity with anbodies of serovar israelensis. While, entomocidus INA288 contained a novel crystal protein gene cryINA288, on plasmid. In addition, the similarity of amino acid sequence between cryINA288 and cry4Aa was 38%. On the other hand, serovar aizawai Bun 1-14 a little homology with serovar entomocidus INA288 ( Figure 2). Quantitative toxicity test isolate The serovar entomocidus INA288, serovar aizawai BUN 1-14 and israelensisONR60A were bioassayed against 4 th -instar larvae of B. mori, P. xylostella, S. litura, A. japonicas, A. aegypti and C. quiquefasciatus. Respectively, serovar entomocidus INA288, aizawai BUN 1-14 showed not toxic activity against latter 3 former lepidoteran species, while serovar entomocidus INA288, aizawaiBUN1-14 and israelensis ONR 60A had the toxicity against latter 3 dipteran species (A. aegypti, A. japonicas and C. quinquefasciatus) ( Table 1). However they had differences form crystal protein, but they had unique as insecticidal to control the same orders (Mosquitoes).

Conclusion
In the search for potential alternatives to the application of B. thuringiensis serovar israelensis ONR60, entomocidus INA288 and aizawai BUN 1-14 isolation of novel mosquitocidal strains. The crystal proteins form of B. thuringiensis serovar entomocidus INA288 has cuboidal shaped, serovar aizawai BUN 1-14 has homology shaped ones and B. thuringiensis serovar israelensis ONR60A has irregular., which was composed of major protein of 130 kDa peptides. B. thuringiensis serovar entomocidus INA288 has 70 kDa and aizawai BUN 1-14 had 69 kDa.