As demand for Tenebrio molitor increases in animal feed market, improvement of mass-rearing indoor system became more crucial. This study is aimed at establishing the technology for conserving genetic resource of Tenebrio molitor, and increasing the technology utilization. This year, in advance, this experiment was designed for examining the developmental characteristics of eggs and larvae. To research the developmental characteristics under different temperatures, eggs, larvae and papae were treated at 17.5, 20, 22.5, 25, 27.5 and 30°C. At 25°C, larvae were exposed to various photoperiod conditions, LD 14:10, 12:12, and 10:14. The results show that the temperature range which affected hatching was 15~32.5°C. To achieve 70 percent or higher hatching rate 17.5~27.5°C was required. Furthermore, hatching occurred within 10 days, when the eggs were treated in 22.5~27.5°C. In regard of larval development, shorter developmental period, and higher pupation and eclosion rates were observed in 25~27.5°C. The shortest larval period was recorded at 14L:10D. At 10L:14D, in contrast, pupae showed the lowest chance of eclosion. Overall, larval development was optimal in long-day condition.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

Bacillus thuringiensis (Bt) is characterized by its ability to synthesize crystal toxins and also able to produce bacteriocins such as thuricin, tochicin, entomocin and bacthuricin. The present work, for the first time, describes the biological activity of bacteriocins from B. thuringiensis subsp. cameroun (Btc). Supernatant which was produced from a liquid culture of Btc had antimicrobial activity against various Bt subspecies, ending up to making a inhibition zone on an agar medium. A significant reduction in antimicrobial activity was observed when the supernatant was exposed to heat at 47~50°C for 15 min. Proteins were separated from the supernatant by a fast protein liquid chromatography (FPLC) given the thermal instability. A group of FPLC fractions had antimicrobial activity against Bt subsp. palmanyolensis, israelensis, 1-3, morrisoni, toguchini and kurstaki and a Bacillus. cereus ATCC21768, ATCC14579 and NRRLB-569. Interestingly, when the supernatant was individually incorporated into the liquid cultures of Bt subsp. israelensis (Bti) and mogi (Btm) with mosquitocidal activity, a vegetative cell growth was observed only in the Btm culture 10 h post-incubation. A possible recovery of vegetative Btm cell growth was observed, compared to a control without the supernatant. These results suggest that Btc produced proteinous antimicrobial substances, one of which may be used as a selection marker to separate Btm after possibly conjugating the two mosquitocidal strains.

Among 154 putative ORFs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), ac78 and ac79 are highly conserved genes in baculovirus, but their functions in the virus life cycle have been unknown so far. To determine their roles in AcMNPV replication, knockout mutants, ac78KO and ac79KO, were constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that both of ac78 and ac79 transcripts were first detected at 6 hours post-infection, and accumulated to maximum at 24 hours post-infection, suggesting that both of ac78 and ac79 are belong to late gene. When the genomic DNA of ac78KO was transfected into Sf9 cells, viral replication was restricted to a single cell infection. These results demonstrated that the ac78 play an important role in BV production, and therefore is essential for AcMNPV to mount a successful infection. Whereas Sf9 cells infected with the ac79KO showed normal viral symptoms such as rounding and swelling, OBs were not observed from majority of infected cells. These results suggested that the ac79 might play an important role in OB production.

Bacillus thuringiensis (Bt) is characterized by its ability to synthesize crystal toxins and also able to produce bacteriocins such as thuricin, tochicin, entomocin and bacthuricin. The present work, for the first time, describes the biological activity of bacteriocins from B. thuringiensis subsp. cameroun (Btc). Supernatant which was produced from a liquid culture of Btc had antimicrobial activity against Bacillus cereus, ending up to making a inhibition zone on an agar medium. A significant reduction in antimicrobial activity was observed when the supernatant was exposed to heat at 75~100°C for 15 min. Proteins were separated from the supernatant by a fast protein liquid chromatography (FPLC) given the thermal instability. A group of FPLC fractions had antimicrobial activity against Bt subsp. palmanyolensis, israelensis, 1-3, morrisoni, toguchini and kurstaki, and B. cereus ACTC21768, ATCC14579 and NRRLB-569. Interestingly, when the supernatant was individually incorporated into the liquid cultures of Bt subsp. israelensis (Bti) and mogi (Btm) with mosquitocidal activity, a vegetative cell growth was observed only in the Btm culture 10 h post-incubation. A possible recovery of vegetative Btm cell growth was observed, compared to a control without the supernatant. These results suggest that Btc produced proteinous antimicrobial substances, one of which may be used as a selection marker to separate Btm after possibly conjugating the two mosquitocidal strains.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

We isolated two baculoviruses, Spodoptera litura granulovirus (SlGV) and S. litura nucleopolyhedrovirus (SlNPV) in the dead larvae of S. litura. The granule of SlGV were ovoidal shape with an approximate measure of 240-340 nm×140-180 nm, and each granule contained one single rod-shape virion with a mean size of 180-200 nm×20-40 nm. Whereas, the polyhedra of SlNPV were irregular in shape with a approximate diameter of 1.0-1.5 ㎛, and numerous virions comprised of the multinucleocapsid were contained in each polyhedra. The major component of occlusion bodies produced by SlGV and SlNPV were about 29 and 30 kDa, respectively. When the phylogenic relationship between these viruses were analyzed using the nucleotide sequences of granulin gene from SlGV and polyhedrin gene from SlNPV, they were not closely related to each other. We also found that the two viruses showed similar insecticidal activity against 2nd instar larvae of Spodotera litura in terms of dose-response, but SlGV showed much longer LT50 than that of SlNPV. The two baculoviruses might be cooperatively be applied as biological control agent for the control of S. litura

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and harbors 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, 55 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells, 16 of them were found affecting viral replication since they defected in producing polyhedra. Furthermore, to verify the pathogenicity of the single ORF-truncated mutants, the remaining 39 mutants were subjected to bioassay to Spodoptera exigua 3rd instar larvae. Among them, ac9-, ac49-, ac103- and ac105-knockout mutants showed higher mortality compared to that of bAc-MK. These results suggested that these ORFs could be related to pathogenicity of AcMNPV.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

A new Bacillus thuringiensis isolate 19-22 (Bt 19-22) exhibited high anti-fungal activity against barley powdery mildew (Blumeria graminis f. sp. hordei). The cry gene content of Bt 19-22 comprised cry1Aa, cry1Ab, cry1Ac and cry1D which have high insecticidal activity against lepidopteran larvae. We tried to confer a dipteran insecticidal activity to Bt 19-22 for constructing a recombinant strain which has multiple functions, anti-fungal and dual insecticidal activity. The insecticidal cry11Aa gene of B. thuringiensis was constructed under cry1Ac promoter in an E. coli-B. thuringiensis shuttle vector (pPro11A). The plasmid, pPro11A was introduced into Bt 19-22 isolate by electroporation and four transformants which had different cry gene contents were identified by PCR with cry11Aa and cry1-type specific primers. Among them, a Bt 19-22 transformant (11A/19-22 No. 7) expressed Cry11A protein (approximately 70 kDa) successfully without change of its inherent characteristics such as Cry protein expression and antifungal activity. The insecticidal activity of 11A/19-22 No. 7 was checked against Plutella xylostella and Culex pipiens. These results suggests that the recombinant strain shows dual insecticidal activity against lepidopteran and dipteran larvae as well as antifungal activity.