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.

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. Until recent times, most of the studies for these fungi have been based on isolation from insect cadaver or soil. However, these methods, especially isolation from soil, might cause a problem involving differential isolation of the each entomopathogenic fungi. The purpose of this study is to determine the optimal isolation medium for entomopathogenic fungi using dodine, chitin, and skim milk. The growth rates of entomopathogenic fungi and non-entomopathogenic fungi were compared on dodine agar medium. The medium for this experiment was modified Veen semiselective medium which consisted of based on SDA (Sabouraund Dextrose Agar), 100 mg/ml for chloramphenicol, 50 mg/ml for streptomycin and adjusted dodine to 40, 50, 70 and 100 mg/ml. As a result, optimal concentration of dodine for isolation of entomopathogenic fungi was 50 mg/ml and 168 positive entomopathogenic fungi were isolated in 470 soil samples and 11 cadavers of insect. In addition, the isolates had significantly greater chitinase and protease activity than non-entomopathogenic fungi. The isolation method described represents a valuable tool for rapid and simple isolation of entomopathogenic fungi. These positive entomopathogenic fungi may have potential against variety pests in agriculture.

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from M. saltuarius cadaver to control it. We collected the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius.