Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. Therefore, this study was performed to select the antimicrobial activity of entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. In addition, we also performed to screening of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity compounds from liquid culture filtrates of entomopathogenic fungi and investigate to it’s anticancer activity. As results, 12 isolates, 6 isolates and 25 isolates showing of these fungal metabolites produced antibacterial, antifungal and radicals scavenging activity compounds, respectively. The preferential antimicrobial and radical scavenging activities give evidence that these entomopathogenic fungal metabolites might be useful as a source for plant pathogen control and pharmaceutical interests.

The bulb mite (Rhizoglyphus echinopus) damages garlic, shallot and onion in the bulbs, corms and tubers. It has recently become a serious problem because of the continuous use of acaricides resulting in resistance among bulb mite population. Thus, there is need to find alternative control measures to suppress bulb mite population. Here, we report the screening result of pathogenic fungi for the control of R. echinopus. Initial screenings were performed using 352 isolates of entomopathogenic fungi from Korea soils. As results, 15 isolates of acaropathogenic fungi showed the pathogenicity to bulb mite supporting fungal conidiation. These isolates were identified as 3 isolates of Metarhizium flavoviride var. pemphigi and 12 isolates of Metarhizium pingshaense by microscopic examination and genetic sequencing of the ITS region and elongation factor-1 alpha. Selected 15 isolates were tested for their virulence against adult R. echinopus and the thermotolerance and the activity to UV-B irradiation of conidia. Additionally, the activities of chitinases and proteases produced by M. pingshaense were compared according to the medium. These acaropathogenic fungi would be considered promising for biological control of bulb mite.

Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. This study was performed to evaluate the antimicrobial activity of 207 entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. As results, twelve isolates (5.7%) and six isolates (2.8%) showing the greatest inhibition against R. solanacearum and B. cinerea, respectively. The culture supernatant of these selected isolates completely suppressed the growth of the pathogen, indicating that suppression was due to the presence of antimicrobial compound in the culture filtrate. The stability test of the culture filtrate showed that the antimicrobial component was heat stable and not protein. These entomopathogenic fungal metabolites may be a good feature to be used in the development of a new biocontrol method of R. solanacearum and B. cinerea.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Hyphantria cunea is a fall webworm is considered an agricultural pest. It is a major pest of many board-leaved trees. H. cunea nucleopolyhedrovirus (HcNPV) and H. cunea granulovirus (HcGV) were isolated from the fall webworm cadavers in Korea. To better understand HcNPV and HcGV, their genomic sequences were determined, analyzed and compared to two viruses together. The entire nucleotide sequence of the HcNPV genome was fully sequenced using 454 pyrosequencing. The genome of the HcNPV was 131,302 bp with a 45 % G+C content. Computer assisted analysis predicted 146 open reading frames (ORFs) of 50 or more amino acids that showed minimal overlap. Further more, when the phylogenetic relationship was analyzed, HcNPV was closely related to Orgyia pseudotsugata MNPV (OpMNPV) which belong to Group I NPV. The HcGV genome was 114,557 bp with a 39% G+C content and contained 130 putative ORFs of 50 or more amino acids. When phylogenetic relationships were analyzed, HcGV was closely related to Xestia c-nigrum granulovirus, which belong to the Type-II GV. HcNPV shares 48 ORFs with HcGV. The most significant difference between HcNPV and HcGV is fgf gene. HcNPV contains one fgf gene, whereas HcGV contains three fgf genes. The presence of fgf reduces the time and efficient systemic infection it takes the virus to kill its host. The difference of fgf number from HcNPV and HcGV suggested that different affect for the speed of systemic infection.

The green peach aphid, Myzus persicae Sulzer, is one of the most important pests affecting protected and open-grown crops, because they cause direct damage by feeding on crops and indirect damage as virus vectors. It has recently become a serious problem because of the continuous use of insecticide resulting in resistance among green peach aphid population. Thus, the development of entomopathogenic fungi as aphid biocontrol agents has received increasing interest as part of integrated control strategies. In this study, we report the screening result of pathogenic fungi for the control of green peach aphid. Initial screenings were performed using 347 isolates of putative pathogenic fungi from Korea soils. As results, 20 isolates of entomopathogenic fungi were isolated from cadavers of green peach aphid supporting fungal conidiation. These isolates were identified as three strains of Lecanicillium attenuatum, nine strains of Beauveria bassiana, one strain of Metarhizium anisopliae, one strain of Metarhizium flavoviride, five strains of Paecilomyces lilacinus, one strain of Aspergillus sp. by microscopic examination, genetic sequencing of the ITS region and Universally Primed PCR (UP-PCR). Based on the screening results, twenty isolates were tested for their pathogenicity against adult green peach aphid. All fungal isolates were pathogenic to green peach aphid but mortality varied with isolates. These entomopathogenic fungi may be useful to develop eco-friendly insecticide to control green peach aphid.

The polyhedrin is responsible to form polyhedra of nucleopolyhedrovirus(NPV) and highly conserved in most completely sequenced in lepidopteran NPVs. Previously, we have reported that the substitution of polyhedrin of Autographa californica NPV(AcNPV) with that of Spodoptera exigua NPV(SeNPV) or Bombyx mori NPV(BmNPV) result the change of polyhedra morphology. In this study, we investigated the influence of changed polyhedra morphology to the virulence of AcNPV. The recombinant AcNPVs were propagated in Spodoptera frugiperda clone 9, 21 cells and S. exigua larvae. Each collected recombinant polyhedra were used in bioassays using S. exigua larvae. The recombinant AcNPVs show that difference virulence according to the polyhedra morphologies. Internal and external morphological features of each recombinant AcNPV were also compared on the electron microscope. Our results suggest that the morphology of polyhedra influence the virulence of NPV and is well worth considering for the development viral insecticide.

The entomopathogenic fungi were an important natural pathogenic of insects that has been developed as potential biological control agents for many important agricultural, forest and medical pests. Several these fungi produce a wide range of secondary metabolites with high therapeutic value as antibiotics, cytotoxic substances, insecticides, compounds that promote or inhibit growth, attractor and repellent. Therefore, this study was performed to select the antibacterial activity of liquid culture filtrates of 347 entomopathogenic fungi form Korea soils against two pathogenic bacteria including Ralstonia solanacearum and Escherichia coli using novel method which represents a quick and easily applicable tool obtaining large number of samples. As results, eight-five strains (24%) and seventy-six strains (22%) of these fungal metabolites produced anti-R. solanacearum and anti-E. coli compounds, respectively. The preferential antibacterial activity against R. solanacearum and E. coli gives evidence that these entomopathogenic fungal metabolites might be useful as an agent for bacteria control and the technique was simple to operate and allowed a large number of samples to be handled concurrently.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

The two-spotted spider mite, Tetranychus urticae Koch, is an economically important pest of crops of plant grown in the field or greenhouse worldwide. It has recently become a serious problem because of the continuous use of acaricides resulting in resistance among spider mite population. Thus, there is a need to find alternative control measures to suppress spider mite populations. In this study, we report the screening result of pathogenic fungi for the control of spider mite. Initial screenings were performed using 352 isolates of putative pathogenic fungi from Korea soils. As results, 11 strains of acaropathogenic fungi were isolated from 8 cadavers of spider mite supporting fungal conidiation. These isolated were identified as four isolates of Beauveria bassiana (6, 2R-3-3-1, 2R-4-5, 2R-4-7), two isolates of Metarhizium anisopliae (4-2, 2-2), one isolate of Clonostachys rosea 5-2, one isolate of Lecanicillium attenuatum 4-1, one isolate of Pochonia suchlasporia 2R-3-1, one isolate of Aspergillus flavus 7 and one isolate of Isaria lilacinus 2R-4-6 by microscopic examination and genetic sequencing of the ITS region. Based on the screening results, eleven isolates were tested for their virulence against adult spider mites. All fungal isolates were pathogenic to spider mite but mortality varied with isolates. These acaropathogenic fungi may be useful to develop eco-friendly acaricide to control two-spotted spider mite.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected Mamestra brassicae (Lepidoptera: Noctuidae) larvae in Korea. Restriction endonuclease fragment analysis using EcoRI, PstI, and BamHI estimated that the total genome size of MabrNPV-K1 is about 150 Kb. The full genome sequences of MabrNPV-K1 were determined, analyzed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,471 bp and had an overall G + C contents of 39.90 %. Computer-assisted analysis predicted 159 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. The gene content and arrangement in MabrNPV-K1 were most similar to those of Mamestra configurata nucleopolyhedrovirus-B (MacoNPV-B), including three polh, p10 and lef-8 gene homologues. The MabrNPV-K1 genome contains four homologous repeat regions (hr1,hr2,hr3,hr4) that account for 3.1% of the genome. The genomic positions of MabrNPV-K1 regions hr1– hr4 are conserved with the genomic positions of MacoNPV-B hr1–hr4. This indicates that the position of MabrNPV–K1 hrs is conserved with regard to both the upstream and downstream genes. Given that hrs share higher similarity within a virus strain than any hrs between species, this evidence further indicates that hrs play a fundamental role in viral life cycle and replication process appears to be tightly linked to functional conservation. The dot plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that MabrNPV-K1 is a Group II NPV that is closely related to MacoNPV but with a distinct genomic organization.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

Mamestra brassicae (cabbage moth) is a common European moth of the order Lepidoptera and the family Noctuidae. The larval stage is highly polyphagous and is known to feed on more than 70 species of host plants from 22 families, including Brassica species, lettuce, onion, potato, pea, tomato and apple. M. brassicae has become a significant pest also in Asia due to the damage caused to agriculturally and economically important Brassica crops. It is difficult to control M. brassicae using chemical insecticide because of its rapid development of resistance. The objective of our study, therefore, was the mass production and formulation of a local strain of M. brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) for the development of viral insecticide to control it. In production efficiency of MabrNPV-K1 using M. brassicae larvae, the mortality of the 3rd instar larvae was 100% when inoculated with 1.0 × 105 PIBs/larva and the yield of MabrNPV-K1 was maximal. Regarding the mortality, yield of polyhedra, inoculation doses and required time, the 1.0 × 104 PIBs/larva at 30°C was determined as optimal conditions producing polyhedra efficiently. To formulate MabrNPV-K1, feeding toxicities of various supplements including spreader and ultraviolet (UV) -protectant were determined. Tinopal UNPA-GX which is UV-protectants was effective for protection of polyhedra from UV and showed the increased mortality when added with 1% concentration. Other supplements did not influence significantly the mortality of MabrNPV-K1. Formulated MabrNPV-K1 with several supplements showed higher pathogencity than un-formulated MabrNPV-K1.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

The diamondback moth, Plutella xylostella is a one of the most important pests of various cruciferous crops and has a geographically wide ranging habitat. The heavy dependence on chemical pesticides has created severe pesticide resistance problems. In recent years, Bacillus thuringiensis product have been widely used for P. xylostella control bus genetic resistance in populations to some B. thuringiensis strains, compounded by cross-resistance to several different B. thuringiensis toxins, has also been identified. Such recent resistance problems serve to emphasize the urgent need for alternative control agents and their use within an integrated pest management approach. Baculoviruses have been used as agents for the biological control of certain insect pest species. the granuloviruses (GVs), based on the structure of the occluded virus and the occlusion body (OB). Several reports have showed P. xylostella granulovirus (PxGV) as a promise control agent for P. xylostella. However, it is very difficult to study GV because its OB, granule, has very small size and could be observed exactly under the electron microscopy (EM). This study was performed to develop rapid quantification method for granule of PxGV. After the exact quantification of granule with latex beads using EM, the universal extraction method of viral DNA was established for consistent experiment. The number of granules was calculated by the quantification of PCR products for granuline gene using spectrophotometer and densitometer. This novel calculation method for granule would be useful to study GV.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Entomopathogenic fungi were isolated directly from a cadaver of adult M. saltuarius (infected with white fungi) supporting fungal sporulation, to develop biological control of pine wilt disease vector, M. saltuarius which was the most abundant in the middle to northern part of Korea and caused enormous damage to native pine tree in Korea, Japan and other regions of Asia. Pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. As the result, only one of them showed pathogenic to adults of M. diphysis, with up to 100% mortality within 13 days of inoculation. Selected fungus was named as MsW1 and identified by Beauveria bassiana using microscopic examination, B. bassiana-specific PCR primers and genetic sequencing of the ITS region analysis. Pathogenicity test were conducted with various concentration of conidial suspensions of this isolate on M. saltuarius (3rd instar larvae and adults). Mortality rates varied from 57.1% to 100.0% and from 16.7% to 100.0% of M. saltuarius (3rd instar larvae and adults), respectively at 30 days. This is the first report of natural infection of M. saltuarius by B. bassiana.