Amaret Bhumiratana

The comparative persistence of toxicity of Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype H-14 against mosquito larvae in different kinds of environments☆

Abstract Bacillus sphaericus strain 1593 and B. thuringiensis serotype H-14 were evaluated for persistence of toxicity against two species of mosquito larvae, Culex quinquefasciatus and Aedes aegypti , in a selected simulating plot in Bangkok. Both strains of bacteria demonstrated larvicidal activity towards both species of mosquito larvae. In tap water, the toxicity of B. sphaericus strain 1593 was found to be greater towards C. quinquefasciatus larvae than A. aegypti larvae, whereas the toxicity of B. thuringiensis serotype H-14 was found to be greater towards A. aegypti larvae than C. quinquefasciatus larvae. The persistence of toxicity of these two bacteria was found to be different. The lethal concentration of B. thuriengiensis H-14 against A. aegypti decreased from LC 90 to below LC 50 in about 15 weeks when tested in tap water. The decrease was faster in polluted water. The toxicity of B. sphaericus 1593 towards C. quinquefasciatus larvae persisted for at least 9 months in tap water and 6 months in polluted water. The multiplication of bacteria was indicated only in populations of B. sphaericus 1593 tested with C. quinquefasciatus larvae.

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura.

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35°C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35°C for 1h. Higher activity was observed in the presence of surfactants, Na(+), NH(4)(+) ions, NaN(3) and ethylenediaminetetraacetic acid (EDTA), while Co(2+) and Cu(2+) ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.

Expression of Chitinase-Encoding Genes in Bacillus thuringiensis and Toxicity of Engineered B. thuringiensis subsp. aizawai Toward Lymantria dispar Larvae

Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl β-D-N,N′- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae (Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.

Community Participation and Appropriate Technologies for Dengue Vector Control at Transmission Foci in Thailand

ABSTRACT A community-based dengue vector control trial was conducted at transmission foci in Plaeng Yao District, Chachoengsao Province, eastern Thailand. Implementation was done by the local community in collaboration with local administration, public health, and school authorities. Our cost-effective approaches combined a source reduction campaign with appropriate vector control technologies applied within the foci (within 100 m around the foci) and also within schools attended by children from the treated areas. Vector management measures by local government included cleanup campaigns before the rainy season followed by a routine garbage pickup during the rainy season. Locally made screen covers for water jars, a combination of local Bacillus thuringiensis subsp. israelensis and Mesocyclops thermocyclopoides (copepod), and locally made lethal ovitraps were appropriate technologies used by the community in this campaign. The success of our intervention was evidenced by the significant reduction of dengue vectors and dengue hemorrhagic fever cases in treated areas compared with untreated areas.

Multiple chitinase enzymes from a single gene of Bacillus licheniformis TP-1

Three chitinase activity bands in a culture supernatant of Bacillus licheniformis TP-1 were detected by non-denaturing PAGE. They were designated chitinase bands 1, 2, and 3 in order from the gel origin. Analysis by immunodiffusion and immunoelectrophoresis using polyclonal antibody raised against chitinase band 3 indicated that these chitinases were antigenically similar. B. licheniformis TP-1 and Escherichia coli harboring the cloned chitinase gene (pCHIL3) from strain TP-1 expressed 3 chitinase bands by non-denaturing PAGE and SDS-PAGE which gave estimated molecular masses of 68, 62, and 50 kDa (named Chi68, Chi62, and Chi50, respectively). All three chitinases had the same N-terminal amino acid sequences, strongly suggesting that Chi62 and Chi50 were derived from Chi68 by a processing step(s) at the C-terminus. The deduced C-terminal amino acid sequence of Chi68 showed homology to amino acid sequences of known chitin and cellulose binding domains. Chi62 and Chi50 lacked this domain (judging from their deduced amino acid sequences and calculated molecular masses) and they were unable to bind chitin, suggesting that they were generated from Chi68 by cleavage of the chitin binding domain at the C-terminus. Comparison of chitinase activities indicated that this binding domain was important for complete hydrolytic activity towards colloidal chitin.

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Abstract Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC50s were 5.6 × 104 and 2.4 × 105 spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Abstract Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K2HPO4 and 4% HDL (H4 medium) or 0.05% K2HPO4 and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC50 value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 108 cells/ml and 10−7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 108 cells/ml and 10−6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC50 value were found to be 1.4 × 109 cells/ml and 10−7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 108 cells/ml and an LC50 value of 10−6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was

Technology transfer for small and medium soy sauce fermentation factories in Thailand : a consortium approach

0.02 and

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium (

Inhibition of a conjugation-like gene transfer process in Bacillus thuringiensis subsp. israelensis by the anti-S-layer protein antibody

7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium (