• The Korean Journal of Pesticide Science
• /
• v.12 no.2
• /
• pp.184-191
• /
• 2008

Entomopathogenic bacteria (Xenorhabdus nematophila, X. sp. and Photorhabdus temperata subsp. temperata) isolated from entomopathogenic nematodes express potent insecticidal activity in insect hemocoel. They are also known to suppress insect immune mediation by inhibiting phospholipase $A_2$, leading to host immunosuppression. This study analyzed effects of their cultured broths on inhibiting insect immunosuppression. For this, we removed all bacterial cells using $0.2\;{\mu}m$ pore sized membrane from the bacteria-cultured broth. All three sterilized cultured media, in dose-dependent manners, significantly inhibited hemocyte-spreading behavior of 5th instar larvae of Spodoptera exigua. However, they showed differential inhibitory activities among different bacterial species, in which X. nematophila showed the most potent inhibitory activity. This immunosuppressive effect was applied to increase the pathogenicity of Bacillus thuringiensis (Bt). All three bacterial cultured broths including bacterial cells significantly potentiated Bt pathogenicity against young S. exigua larvae when each of them was orally administered in a mixture of low dose of Bt. Finally, we tested the effect of oral administration of the cultured media containing the immunosuppressive compound(s) secreted by the bacteria. The membrane-sterilized cultured broths were mixed with the low dose of Bt and then orally administered to the young S. exigua. Only the cultured medium of X. nematophila showed increase of Bt pathogenicity. These results indicated that the; cultured media of the three bacteria possessed immunosuppressive factor(s), which may act to potentiate Bt toxicity to young S. exigua larvae.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.9
• /
• pp.937-946
• /
• 2011

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on $LC_{50}$ values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.507-521
• /
• 2014

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared with a wild-type primary form, a secondary form usually loses several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in its production of eicosanoid-biosynthesis inhibitors (EBIs) compared with the primary form of X. nematophila. Production of oxindole and p-hydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila enhanced the pathogenicity of Bacillus thuringiensis ( Bt) significantly more than the culture broth of the secondary form. Furthermore, this study developed a highly efficient "Dual Bt-Plus: to control both lepidopteran insect pests Plutella xylostella and Spodoptera exigua, by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.

• Korean journal of applied entomology
• /
• v.51 no.1
• /
• pp.39-45
• /
• 2012

The entomopathogenic bacterium, $Xenorhabdus$ sp., was isolated from an entomopathogenic nematode, $Steinernema$ $monticolum$. When these bacteria were injected into the hemocoel of the diamondback moth, $Plutella$ $xylostella$, they caused significant mortality. However, the bacterium was not pathogenic when it was administered orally. This study showed that $Xenorhabdus$ sp. significantly enhanced oral pathogenicity of $Bacillus$ $thuringiensis$ (Bt) against the last instar larvae of $P.$ $xylostella$. Different ratios of culture broth of $Xenorhabdus$ sp. and Bt showed significantly different pathogenicities against $P.$ $xylostella$. In field tests, the optimal bacterial mixture significantly enhanced control efficacy against $P.$ $xylostella$ compared to Bt treatment alone. These results demonstrated that $Xenorhabdus$ sp. culture broth can be developed as a potent biopesticide by enhancing the insecticidal efficacy of Bt.

• The Journal of Korean Society of Virology
• /
• v.29 no.3
• /
• pp.145-153
• /
• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.

• Korean journal of applied entomology
• /
• v.43 no.1
• /
• pp.27-34
• /
• 2004

This study was conducted to develop optimal control tactics of the northern root-knot nematode, Meloidogyne hapla, using cultural method and biological agents ｛Bacillus thuringiensis (Bt), Paecilomyces lilacinus and plant extract (Huhjunl)｝ in the fields of Codonopsis lanceolata. Germination of C. lanceolata was susceptible to fosthiazate, but not to Bt or a plant extract. In pot assay, the inhibitory effect of two microbial agents, Bt and Paecilomyces lilacinus, on M. hapla were significant, but less than that of fosthiazate. The plant extract also had significantly inhibitory effect on M. hapla. In field assay, treatments of P lilacinus and fosthiazate resulted in maximal yields and qualities of C. lanceolata. The effect of the plant extract on the yields of C. lanceolata was also better than no treatment. The nematode-occurring condition of the fields before transplanting had significant effect on development of C. lanceolata; nematode-occurring field type gave less yields than nematode-free field type. These results suggest that a cultural control technique using paddy field, microbial pesticides using Bt or P lilacinus, and the plant extract are the promising control tactics against M. hapla in C. lanceolata fields. As a field manual to decrease economical damage of C. lanceolata due to M. hapla, this study suggests that C. lanceolata can be cultured directly in paddy field or in upland field after nematode control using microbial agents or the plant extract.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.17 no.1
• /
• pp.11-18
• /
• 1997

This study was to provide the basic data in improving protoplast formation and regeneration of antagonistic bacteria against phytopathogenic fungi and pest. The antagonistic rhizobacterium, BS 101, against Rhizoctonia solrmi and Fusurium oxyspomm was isolated and identified as Bacillus subtilis. Another bacterium for protoplast formation and regeneration was B. thuringiensis subsp. kurstcJtiHD-l (BT 37669) which have insectcidal toxin in the orders Coleopteria, Dipteria etc.. Auxotrophic mutants, BS 1013 and BT 69, were isolated by treating with NTG 300 ug/ml for 40 min. at $37^{\circ}C$, and with NTG 300 ug/ml for 30 min. at $37^{\circ}C$, respectively. The BS 1013 and BT 69 were converted to protoplas by treating with lysozyme 300 ugh1 for 30 min. at 37C, and lysozyme 9 mglml for 60 min. at $37^{\circ}C$, respectively. The fequencies of the protoplast formation of BS 1013 and BT 69 were 90.00 and 92.83% respectively, after 1~2 day at $37^{\circ}C$. The regeneration kequencies of the protoplasts BS 1013 and B T 69 were 0.52 and 0.10%, respectively, after 4~6 days at $37^{\circ}C$.

• BMB Reports
• /
• v.47 no.10
• /
• pp.546-551
• /
• 2014

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae.

• Microbiology and Biotechnology Letters
• /
• v.17 no.4
• /
• pp.295-300
• /
• 1989

Screening of Pseudomonas strains that can be used as hosts for expression of crystal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 was carried out. From rhizosphere soil of 7 kinds or crops as fluorescent Pseudomonas strains were isolated. A hybrie plasmid, pKTC1, composed of the broad host range vector pKT230 and the crystal protein gene was constructed and used for transformation of the 35 Pseudomonas strains. As the result, the crystal protein gene could be introduced into 4 isolates. Several methods including bioassay and immunochemical detection indicated that the crystall protein gene was expressed in the Pseudomonus isolates.

• Journal of Microbiology and Biotechnology
• /
• v.1 no.1
• /
• pp.37-44
• /
• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.