Proceeding of 2013 International Seminar on Climate Change and Food Security ISCCFS 2013
Palembang, South Sumatra -Indonesia, 24-25 October,2013
170
2. Methodology
2.1. Isolation of
B. thuringiensis
Five g of soil samples is diluted well in 15 ml dH
2
0 in test tube. Shaked well until perfectly diluted. One ml of upper part of dilution is taken in eppendorf tube, added by 1 µl Triton X-100, and heated in water bath
85ºC 15 minutes. With a sterile spatula, the solution was streaked on the medium NaCl Glycine Kim and Goepfert NGKG on petridish. Petridish was incubated at 30ºC, for 24-72 hours. Colonies of B.
thuringiensis will grow in white color. After 24-72 hours incubation, proteinaceous parasporal inclusion bodies will presence. Identification of
B. thuringiensis
refers to Thiery and Frachon 1997, by microscopic observation of bacterial cells tests, gram staining.
2.2. Production of crystal protein, SDS-PAGE and SEM
Propagation of B. thuringiensis using Luria Bertani medium LB, 5 g yeast extract, 10 g Tryptone and 10 g NaCl in 1000 ml volume, pH 7.2 and sterilized by autoclave for 20 minutes, the temperature of 121°C 1
atm. One colony of B. thuringiensis than for oblique performed in laminar air flow is taken to be multiplied by 50 ml of LB media and shaken for 2 days with a speed of 150 rpm. Harvesting is done by rotation protein
centrifuge at 2000 rpm for 15 minutes and will be done 2 times. Results of sediment were taken and diluted with 1 ml dH
2
O shaken until homogeneous and stored in Eppendorf tubes and ready for testing.Crystal proteins isolated from B. thuringiensis strains are analyzed by sodium dodecyl polyacrilamide gel
electrophoresis SDS-PAGE.
2.3. Insect test preparation
Groups of eggs of armyworm
S. litura
were obtained from the field and subsequently maintained in the laboratory. Larvae reared in a plastic container maintenance d = 15 cm and h = 9 cm. Food used were the
leaves of water spinach
Ipomoea reptana
grown without pesticide treatment for mass rearing for
S. litura
. Temperature and relative humidity were maintained. Maintenance of container was done by cleaning of
residual dirt and food remains to ensure the availability of food and cleanliness. At the bottom of the box was placed maintenance of sterile soil that had been sterilized as a place of
S. litura
to become pupae. If the larvae has reached pre-pupa phase characterized by no activity, meaning caterpillar will enter the pupa stage.
Larvae of
S. litura
reared to be a phase of insect pupae, and imago. Insect samples used were second generation F2. P. japonica was kindly provided by Dr. Asano Lab of Applied Molecular Entomology,
Hokkaido University, Japan.
2.4. Bioassay of insects
Protein was diluted in sterilized water, smeared on the surface of spinach leaf and given to larva 3
rd
instar of
S. litura
. While treatment to
P. japonica
was done by mixing protein with living medium soil, and 5 individues of 1
st
instar larvae were prepared on thi medium. Mortality of larvae was observed during 7 days after treatment.
3. Results and Discussion
3.1. Isolation of
B. thuringiensis
strains
Sample of soil was prepared from swamp land. Results of isolation found 15 strains of 27 soil samples were positive of B. thuringiensis. To investigate their toxicity against insect pests, it was conducted
screening test with 10
6
sporesml in each strain. The result showed that strain MSP-02 showed very toxic to S
. litura
unpublished data. Therefore it was chosen as an object to find out other characteristic.
3.2. Scanning Electron Microscope result
Cell and protein of
B. thuringiensis
strains were shown in Fig. 1. Cell form was very clear, while the crystal protein was slight and round. It was assumed that this protein caused mortality to insect pest.
Proceeding of 2013 International Seminar on Climate Change and Food Security ISCCFS 2013
Palembang, South Sumatra -Indonesia, 24-25 October,2013
171
Notes: 1: cell, 2: protein
Fig. 1: Profil cell and protein of
Bacillus thuringiensis
MSP-02 strain
3.3. Molecular weight of
B. thuringiensis
crystal protein
5. Fig. 2: Profile of crystal protein of
B. thuringiensis
MSP-02 strain lane 1, SMR-03 strain lane 2 and Protein Size Marker lane 3
Molecular weight of
B. thuringiensis
MSP-02 strain tend to show 135 kDa of molecular weight. As it was a preliminary result, it was assumed that this strain content of Cry I protein as reported by Hoftey
Whitley 1989. The group of Cry I protein was reported toxic to Lepidopteran larvae. The other groups fo example Cry III was toxic only to Coleopteran larvae Cannon, 1995. To prove this statement, it was
conducted bioassay of protein of
B. thuringiensis
MSP-02 strain against
S. litura
and
P. japonica
.
3.4. Bioassay of insect pests
Bioassay was done by : Protein was diluted in sterilized water, smeared on the surface of spinach leaf and given to larva 3
rd
instar of
S. litura
. Treatment to
P. japonica
was done by mixing protein with living medium of this insects soil. The result showed that
P. japonica
was died two of three individues. It was preliminary bioassay, since the number of
P. japonica
was limited Fig. 3.. It was not a good result, because - compared with other strain i.e. SMR-03- only 66,7 were died. There was specificity toxicity of
B. thuringiensis
especially because of content of crystal protein and insect target. As stated by Hoftey Whitley 1989, to be toxic, protein must be digested as protoxin in available mid gut condition. Lepidopteran larvae
possessed alkaline condition pH 9. This condition supported alteration from protein to be protoxin.
1 3
2
kDa 200
116 97
66 45
23
1 2
116 135
Proceeding of 2013 International Seminar on Climate Change and Food Security ISCCFS 2013
Palembang, South Sumatra -Indonesia, 24-25 October,2013
172 Fig. 3: Mortality of
Popillia japonica
by
B. thuringiensis
MSP-02 strain No.2, SMR-03 strain 1, PIK-01 strain No. 3 and PDIK strain No 4
4. Conclusion
