Methyl bromide (MB) has been still routinely used in quarantine fumigation on imported citrus, although there had been issuing chronic inhalation toxicity to fumigators and related workers as well as phytotoxic damages after fumigation. Ethyl formate (EF), is the only option to replace MB in terms of its safety for consumers (food additive and naturally occurred) and worker with higher threshold level limit (TLV = 100 ppm). Its application technology also provide cost effectiveness, good commercial practice in terms of application time (< 10 min) for 40 ft container. The replacement of MB with EF is recommended not to fumigate with hazardous and phasing-out MB on imported oranges.

Carbonyl sulfide(COS) is a naturally generated gas from fermentation process of microbes and from plant root and stem. COS was firstly registered as a fumigant at 1993 to control stored product pests. To supplement environmental problems and toxicity of commercial fumigants and develop new fumigant, we have processed the susceptibility assessment of carbonyl sulfide on important agricultural pests, Myzus persicae and Tetranychus urticae. Every growth stages of two insect species were tested, and five dosages of carbonyl sulfide were treated for 4 hours, and the mortality was investigated after 24 hours of treatment. Nymphal stage of M. persicae was completely controlled at more than 20 mg/L dosage, and adult stage showed 95.8% mortality at 80 mg/L dosage. The LC50 of M. persicae was 7.314mg/L for nymph and 26.117mg/L for adult stage. Egg stage of T. urticae showed 91.2% mortality when treated with 100mg/L carbonyl sulfide, and nymph and adult stage showed 100% and 94.1% mortality at 8mg/L and 80mg/L, respectively. The LC50 of T. urticae was 73.110mg/L for egg, 2.818mg/L for nymph and 12.054mg/L for adult stage.

Stored grain pests can cause reduction of grain quantity, quality, commercial value and germination rate. Susceptibility of three fumigants, methyl bromide, ethyl formate and phosphine, were assessed on Tribolium castaneum, which is an important stored grain pest. On susceptible insects, LCT50 of phosphine was 0.654mg h/L for egg, 0.127mg h/L for late larvae, 0.105mg h/L for pupae and 0.048mg h/L for adult stage, respectively. LCT50 of methyl bromide was 33.193mg h/L for egg, 14.585mg h/L for late larvae, 8.616mg h/L for pupae and 11.967mg h/L for adult stage, respectively. LCT50 of ethyl formate were 25.165mg h/L for egg, 80.912mg h/L for late larvae, 176.326mg h/L for pupae and 68.578mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine were 82.325mg h/L for egg, 33.315mg h/L for late larvae, 73.546mg h/L for pupae and 55.707mg h/L for adult stage, respectively. LCT50 of methyl bromide were 19.250mg h/L for egg, 43.413mg h/L for late larvae, 76.842mg h/L for pupae and 19.387mg h/L for adult stage, respectively. LCT50 of ethyl formate were 87.552mg h/L for egg, 113.457mg h/L for late larvae, 200.122mg h/L for pupae and 85.394mg h/L for adult stage, respectively.

We assessed the susceptibility of three fumigants on Phthorimaea operculella, which is an important pest of stored potato worldwide. 5 to 6 initial dosage of each fumigants were treated on every growth stages of P. operculella. Methyl bromide showed 100% mortality at CT 33.40mg h/ L on egg, CT 14.41mg h/L on late larvae, CT 31.89mg h/L on pupae and CT 16.01mg h/L on adult, respectively. The LCT50 of methyl bromide was 19.115mg h/L on egg, 3.934mg h/L on late larvae, 13.810mg h/L on pupae and 6.260mg h/L on adult, respectively. In case of phosphine, 98% mortality was achieved at CT 16.77mg h/L on egg, and 100% mortality was achieved at CT 16.58mg h/L on late larvae, CT 18.54mg h/L on pupae and CT 12.28mg h/L on adult, respectively. The LCT50 of phosphine was 1.457mg h/L on egg, 2.236mg h/L on late larvae, 1.282 mg h/L on pupae and 0.253mg h/L on adult, respectively. In case of ethyl formate, 100% mortality was achieved at CT 96.21mg h/L on egg, CT 101.30mg h/L on late larvae, CT 120.66mg h/L on pupae and CT 148.30mg h/L on adult, respectively. The LCT50 of ethyl formate was 23.730mg h/L on egg, 13.706 mg h/L on late larvae, 29.578mg h/L on pupae and 19.235mg h/L on adult, respectively.

The existing ethyl formate fumigant is carbon dioxide (CO2) mixed liquified gas in metal cylinder, but this product type costs a lot to manufacture, translate and maintain cylinder. To supplement these problems, we have developed a new ethyl formate fumigation technique with nitrogen (N2) carrier. We assessed the susceptibility of mealy bugs, the most frequently detected pests in imported banana, and phytotoxicity of banana fruits. Ethyl formate and nitrogen were concurrently treated on citrus mealybug, one of the most resistant mealybug to fumigant, and ethyl formate was treated with LC50 product of independent treatment dosage. Nitrogen was treated with 7 dosages from 79% to 95% concentration. Phytotoxicity of banana was assessed by treating EF 35 mg/L with N2 79% for 14 days, and color, sugar contents and loss of weight were measured. EF with N2 treatment showed more than 50% of mortality on every growth stages, and there was no significant difference between control and treatment banana fruits. These results indicate that concurrent treatment of EF and N2 can be used to control mealybug in banana fruits.

Recently, phosphine resistance of Sitophilus oryzae has been reported from China, India, Brazil and Australia. In this study, susceptibility of three fumigants were assesses on phosphine resistant and susceptible S. oryzae to investigate domestic phosphine resistance level and to use base data for resistance control. On susceptible insects, LCT50 of phosphine was 0.440mg h/L for egg, 0.602mg h/L for early larvae, 3.901mg h/L for late larvae, 6.171mg h/L for pupae and 0.295mg h/L for adult stage, respectively. LCT50 of methyl bromide was 9.997mg h/ L for egg, 12.113mg h/L for early larvae, 18.952mg h/L for late larvae, 21.104mg h/L for pupae and 17.824mg h/L for adult stage, respectively. LCT50 of ethyl formate was 75.795mg h/L for egg, 60.110mg h/L for early larvae, 160.491mg h/L for late larvae, 255.797mg h/L for pupae and 77.711mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine was 6.959mg h/L for egg, 28.456mg h/L for early larvae, 48.170mg h/L for late larvae, 29.106mg h/L for pupae and 16.550mg h/L for adult stage, respectively. LCT50 product of methyl bromide was 17.842mg h/L for egg, 14.900mg h/L for early larvae, 25.840mg h/L for late larvae, 43.520mg h/L for pupae and 16.397mg h/ L for adult stage, respectively. LCT50 of ethyl formate was 60.034mg h/L for egg, 64.450mg h/L for early larvae, 149.028mg h/L for late larvae, 140.408mg h/L for pupae and 66.043mg h/L for adult stage, respectively. Domestic resistant S. oryzae showed 4 to 56 times higher resistance rate than susceptible insects.

Phosphine (PH3) resistance in the stored-products insect pests has been reported throughout the world in various insect species, including Rhyzopertha dominica, Tribolium castaneum, and Cryptolestes ferrugineus, leading farmers and fumigators to identify new fumigation tools to control PH3-resistant insect pests in storage facilities. Understanding PH3-resistance mechanisms in insects might contribute to providing clues for the development of new chemicals, including fumigants, to control various PH3-resistant insects. A proteomic study has shown 15 decreased proteins in the PH3-resistant R. dominica (CRD343 strain) in comparison to the PH3-susceptible R. dominica, and among those 15 proteins, dihydrolipoamide dehydrogenase (DLD), a protein involved in the Krebs cycle, was identified (Park et al., 2008). The DLD polymorphisms responsible for genetic resistance have disulfide active sites for PH3 binding and are highly sensitive to arsenic exposure after mutagenesis in insects (R. dominica and T. castaneum) and Caenorhabditis elegans (Schlipalius et al., 2012). Here, two PH3- resistant S. oryzae strains were used to understand the development of PH3 resistance in these insects. Acute toxicity test by PH3 on the two PH3-resistant strains was undertaken followed by ethyl formate inhibition study on cytochrome c oxidase activity. The Lineweaver-Burk plots after inhibition studies showed there were significantly difference in inhibition mode between the resistant strains and the control. The RT-qPCR analysis and the next-generation sequencing of the mitochondrial DNA revealed significant changes in metabolism and energy production. Taken together, the PH3 resistance in S. oryzae was definitely acquired by the overall transformation of biochemical reactions to overcome PH3 toxicity.

Carbonyl sulfide is a chemical that is registered as a crop protection product in Australia due to microorganisms such as soil, plant roots, branches, fertilizers and compost. In order to develop new fumigants for domestic use, the susceptibility of Carbonyl sulfide fumigants to two agricultural pests was evaluated. The susceptibility of peach aphid and spotted mite, which is an agricultural pest, was evaluated. After 5 hours of treatment (10, 20, 40, 60, and 80 mg / L) . As a result, peach aphid larvae showed a high mortality rate of 97.6% at 10 mg / L and 100% at 20 mg / L or more. Larvae were found to have a rate of 95.8% at 80 mg / L. The insect mortality rate was 91.8% at 60 mg / L and 94.1% at 80 mg / L.

The susceptibility of the Cigarette beetle, which are pests in tobacco, to methyl bromide and phosphine fumigants was evaluated. Five concentrations of each methyl bromide were selected for all stages and treated for 4 hours. As a result, 100% larvae were observed in eggs CT 51.20, late larva CT 73.61, pupa CT 71.87 and adult 52.87 mg h / L, respectively. The LCT50 values for methyl bromide were 13.896 for eggs, 36.038 for late larvae, 25.172 and 21.758 mg / l, respectively. The phosphine treatment was carried out for 5 to 6 concentrations (0.025, 0.051, 0.099, 0.501, 0.999 and 1.500 mg / L) for 20 hours. As a result, egg and late larvae showed 100% larvae at CT 5.137 and 6.435 mg h / L, respectively, and 61.9% and 98.9% of larvae and adult larvae were similar to each other. Pupae showed 86.4% larvae at CT 10.520 and 100% larvae at over 13.777 mg / h of CT. The LCT50 values for phosphine were 0.317 for eggs, 0.649 for late larvae, 3.748 for pupa and 0.703 mg / l for adults.

Ethyl formate has been used for the control of insect pests by fumigation. However, there were not many reports toshow its target site of fumigant toxicity on insect pests since its first use in the agricultural industry. In the present study,we showed the presumable target sites of ethyl formate fumigation in insect pests using Myzus persicae nymphs. Afterethyl formate fumigation, the nymphs of this species were collected and the changes at the biochemical and molecularlevel were determined. The activity of cytochrome c oxidase (COX) was approximately two-fold higher after ethyl formatefumigation. In addition, the expression levels of acetylcholinesterase (AChE) decreased gradually with increasing ethylformate concentration. These two findings suggested that COX and AChE might be the major target sites of ethyl formatefumigation. In addition to these results, the analysis of lipid content using MALDI-TOF MS/MS identified 9 phospholipidsdifferently generated 2-fold higher in the ethyl formate-treated nymphs than that in the control nymphs, thereby leadingto changes in cell membrane composition in M. persicae nymphs. Therefore, the ethyl formate fumigation caused lethaleffects on M. persicae nymphs by changing COX activity, AChE gene expression, and phospholipid production.

Some Japonica rice plant varieties show the resistance to the whitebacked planthopper (S. furcifera) and the varieties produce ovicidal compound, benzyl benzoate, to kill their eggs lying in the rice plant. We have tried to elucidate the mechanism for inducing the production of the benzyl benzoate by the Japonica rice varieties. The active 80% MeOH/H2O extract was chromatographed on ODS open column and separated into 6 fractions; 100% H2O, 20% MeOH/H2O, 60% MeOH/H2O, 80% MeOH/H2O, 100% MeOH, 100% EtOH. Of these 6fractions, 100% MeOH and 100% EtOH fractions evidently induced benzyl benzoate at 43.95μg/g of fresh rice plant(frp) and 31.04μg/gfrp, respectively. The active compounds were submitted to analyses by using NMR and GC- and LC-MS inorder to elucidate the structure. We find elicitors to induce with S. furcifera. The elicitors are in the female of S. furcifera and the structures of elicitors are species of phospholipid and glycerol.

Background : Korean ginseng is an important cash crop in Asian countries. However, plant yield is reduced by pathogens. Among the Ilyonectria radicicola-species complex, I. mors-panacis is responsible for root-rot and replant failure of ginseng in Asia. It is essential to find a way to reveal the existence of the pathogen before starting cultivation. Therefore, qRT-PCR method is developed to detect and quantify the pathogen in ginseng soils. Methods and Results : In this study, species specific Histone H3 primer set is developed for the quantification of I. mors-panacis. The primer set was applied on DNA of other microbes to evaluate its sensitivity and selectivity on I. mors-panacis DNA. Sterilized soil samples artificially infected by the pathogen in different concentrations were used to evaluate the ability of the primer set to detect the pathogen population in the soil DNA. Finally, the pathogen was quantified in many natural soil samples. The designed primer set was found to be sensitive and selective to I. mors-panacis DNA. In artificially infected sterilized soil samples, the estimated template using qRT-PCR was positively correlated with the pathogen concentration in soil samples (R2=0.94), disease severity index (R2=0.99), and colony forming unit (R2=0.87). In the natural soils, the pathogen was recorded in the most of fields produce bad yields with the range of 5.82 ± 2.35 to 892.34 ± 103.70 pg/g of soil. Conclusion : According to the presented results, the proposed primer set is applicable for estimating soils quality before ginseng cultivation. This will help in the disease management and crop protection in the future.

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.