Chrysanthemum stunt viroid (CSVd) is a serious pathogen affecting chrysanthemum that has caused significant economic losses to Chrysanthemum flower production worldwide. Control of CSVd disease is difficult due to its contagious nature and long latent period in the field. As chrysanthemum is most often produced by implanting seedlings, it is necessary to diagnose CSVd infection before cultivation. In this study, we screened CSVd infection in seedlings from 30 varieties including 5 domestic, 6 Japanese, and 19 European varieties. Molecular diagnosis of the combination of RT-PCR and nested PCR showed that CSVd was not detected by the first RT-PCR but detected by the second nested PCR analysis in 10 varieties, including 1 domestic, 2 Japanese, and 7 European varieties. Further comparison of 10 identified CSVd nucleotide sequences showed that those are highly conserved (99-100%) and the most similar to an isolate (AB006737) identified in Hokkaido, Japan. Our study suggests that the combination of RT-PCR and nested PCR analysis is successful for the CSVd diagnosis of seedlings and the molecular diagnosis is necessary to prevent the introduction and propagation of viroid disease into the fields.

Diapause duration of Paratlanticus ussuriensis is prolonged as an egg that enter both initial and final diapause stgaes. Environmental conditions, such as temperature, can modify the duration of initial diapause. Eggs enter initial diapause at 20℃, but continued early embryonic development at 30℃. Final diapause at a fully developed embryonic stage is obligatory regardless of temperature conditions. To determine temperature effects on initial diapause mechanism of P. ussuriensis eggs, we compared weights, DNA and RNA amounts of eggs incubated at either 20℃ or 30℃ for 50 days after oviposition. We identified small heat shock protein (shsp), heat shock protein 90 (hsp90) and three heat shock protein 70 (hsp70a, hap70b, hsp70c) genes of P. ussuriensis and determined those expression levels at different temperature conditions. The levels of shsp, hsp70a, hsp70b and hsp90 was not detectable until 20 days after oviposition at both temperature conditions, but highly increased at 50 and 60 days when incubated at 30℃. In contrast, hsp70c level was rapidly peaked at 20 days after oviposition, which is the time of initial diapause entrance. We analysis of temperature sensitivity of P. ussuriensis eggs. Hsp70a is expressed after the first cold treatment of mature eggs. Hsp70b is highly expressed just before hatching. Both shsp and hsp70c was highly expressed at the heat shock condition into immature egg stage. Our results suggest that high temperature breakdown initial diapause and one hsp gene, such as hsp70c, may be involved into the mechanism of initial diapause of P. ussuriensis eggs.

Paratlanticus ussuriensis enter prolonged diapause at an egg stage. Environmental conditions, such as temperature, can modify the diapause duration at initial diapause. Eggs enter initial diapause at 20℃, but continued early embryonic development at 30℃. Final diapause at a fully developed embryonic stage is obligatory regardless of temperature conditions. To determine temperature effects on diapause mechanism of P. ussuriensis eggs, we compared weights, DNA and RNA amounts of eggs incubated at either 20℃ or 30℃ for 50 days after oviposition. Both egg weight and total amount of DNA were constant at 20℃ but gradually increased at 30℃. However, total RNA level was rapidly increased at 15 days-old eggs at 30℃ and maintained high levels during further period whereas its level was constant at 20℃. In addition, we identified three heat shock protein 70 (hsp70a, hap70b, hsp70c) genes of P. ussuriensis and determined those expression levels at different temperature conditions. The levels of hsp70a and hsp70b was not detectable until 20 days after oviposition at both temperature conditions, but highly increased at 50 and 60 days when incubated at 30℃. In contrast, hsp70c level was rapidly peaked at 20 days after oviposition, which is the time of initial diapause entrance. Our results suggest that high temperature breakdown initial diapause and a certain hsp gene, such as hsp70c, may involve in the initial diapause mechanism of P. ussuriensis eggs.

The diapause duration P. ussuriensis eggs is known to either one year or prolonged to additional years. This complex life cycle is mainly caused by the interruption at two embryonic stages: early stage (initial diapause) and fully developed late stage (final diapause). The environmental cues, such as temperature, is critical to determine the diapause duration but the precise diapause mechanism of P. ussuriensis eggs is unclear. We demonstrated temperature effects on the entrance of initial diapause. Newly oviposited eggs were incubated at two temperature conditions (20℃and 30℃) for 60 days. When eggs were incubated at 20℃, egg weights were not significantly changed, but at 30℃ those were gradually increased to 1.5 times for 50 days. Genomic DNA contents of eggs were similarly increased at 30℃. Total RNA contents at 30℃ were highly increased from the 15-days-old but not at 20℃. In addition, we further analysed expression levels of two heat shock protein 70 (hsp70a, hap70b) genes during embryonic development. Our results suggest that initial diapause occurred between 10 and 15 days after oviposition and its entrance was blocked by high temperature incubation.

Spider mites are one of major pests in cultivations of various ornamental plants and also important in plant quarantine service. Due to the very small body size and high similarity within the Genus the identification of species is difficult even at the microscopic observation. To identify 5 major species (Tetranychus cinnabarinus, T. urticae, T. phaselus, T. kanzawai and T. truncatus) in the Genus Tetranychus at the molecular level, we designed 4 species-specific primer sets using nucleotide sequences of the internal transcribed spacer (ITS2) region in the nuclear ribosomal repeat unit. At the PCR diagnosis of extracted genomic DNAs of 5 species using each primer set, specific primers of both T. phaselus and T. truncates were species-specific to their own species samples. However, specific primer set of T. urticae detected T. cinnabarinus as well as T. urticae. Specific primer set of T. kanzawai detected T. truncates as well as T. kanzawai, even though detection intensities were lower in non-target species.

Oral toxicities of 5 Photorhabdus temperata ssp. temperata (Ptt) strains collected in different regions of Korea were determined against the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, Culex pipiens pallens and Paratlanticus ussuriensis. When a diet or water containing culture media of 5 different Ptt strains were ingested to immature insects, mortalities of the first instar larvae of G. mellonella, L. caesar, P. ussuriensis and young nymphs of C. pipiens pallens were rapidly increased and 100% within 3-5 days after treatments. However, mortality of P. interpunctella neonate larvae was slightly slower and 94.4-100% within 7 days after treatments. As controls, a diet containing either water, the medium without culturing bacteria, or E. coli culture medium did not effective on their mortalities. As another control group, the culture medium of P. temperata ssp. laumondii (KACC) were variously effective to mortalities of 4 species, namely, 100, 45.3, 2.8 and 0% to Galleria, Lucilia, Plodia and Culex, respectively. Culture media of Ptt strains inhibited developmental late of late larvae of P. interpunctella. Our results suggest that the oral administration of the culture medium of Ptt symbiotic bacteria was highly effective to control various immature insects.

During the last several years, the population of the ussur brown katydid, Paratlanticus ussuriensis has been highly increased at mountain-side orchards in Youngdong province in Korea. Nymphs and adults emerged one time per year from early May to August and cause a serious demage on the crop production. Adults oviposit eggs from July to September under the ground and eggs overwinter at diapause stage. However, the diapause duration P. ussuriensis is known to either one year or prolonged to additional years. The environmental cues, such as temperature, could be a critical to determine the diapause duration but the precise diapause mechanism of P. ussuriensis eggs is unclear. We demonstrated to determine the stage of diapause initiation during embryogenesis. Newly oviposited eggs were incubated for 60 days at two temperature conditions (20℃and 30℃). When the eggs were incubated at 20℃ those weights were not changed, but when they incubated at 30℃ those were increased from 15-days-old. Total RNA contents of eggs incubated at 30℃ were highly increased from the 15-days-old when they compared with those of eggs incubated at 20℃. In addition, total DNA contents of eggs were greatly higher when they incubated at 30℃. Our results suggest that early embryonic development was sensitive to high temperature and it was stimulated at 15-days-old. To determine the temperature response of embryonic development at molecular level we identified heat shock protein 70 (hsp70) gene of P. ussuriensis. Further analysis would conduct the gene expression rate of diapausing eggs on different temperature conditions by using real time RT-PCR technique and determine the molecular characteristics of P. ussuriensis egg diapause.

The use of electron beam irradiation has been emerged as one of alternative ways of the restriction of methyl bromide usage for the disinfestation of stored-products and quarantine pests. Here we demonstrated effects of electron beam irradiation on development and gene expression of P. interpunctella, which is a serious pest of various stored-products. P. interpunctella at various developmental stages were irradiated at 0, 0.25, 0.5, 0.75 and 1 kGy. Eggs failed to hatch at all treated doses of electron beam. Fifth instar larvae pupated only 6.7% by 0.25 kGy irradiation but failed pupation at above doses. Interestingly, survived larvae by low-dose irradiation did not pupated until 40 days. Pupae eclosed to adults only 12.1% by 0.25 kGy irradiation but failed at above doses. In addition, 5-day-old pupae eclosed 94.4, 91.6, 100 and 49.9% at 0.25, 0.5, 0.75 and 1.0 kGy, respectively. However, most of those emerged adults were malformed, especially in wings, and showed very low oviposition rate. We demonstrated whether the electron beam irradiation induces gene expression. Upregulated genes at any doses were hsp70, which is a stress-responsive protein, at fifth instar larval stage and hemolin, which is an immune-responsive protein, at pupal stage. Some genes of pupae, such as β-1,3 glucan recognition protein, hsp70 and acp25 (small hsp) were upregulated only at high doses. However, other genes, such as prophenoloxidase, ultraspiracle , ecdysone receptor and heat shock proteins (hsp90, hsc70) were downregulated by irradiation of electron beam

A glucose-regulated protein 78 (grp78) gene, which is belongs to a heat shock cognate 70 (hsc70) subfamily, was cloned from Indian meal moth, Plodia interpunctella. Its full length cDNA was 2679 bp and contains a 1980 bp open reading frame. The translated amino acid sequence consists of 660 residues with a calculated molecular mass of 72,975 Da and an isoelectronic point (pI) of 5.27. It contains several highly conserved functional motifs of the Hsp70 family and, particularly, C-terminal motif of KDEL that is characteristic for endoplasmic reticulum (ER) hsc70. Its deduced amino acid sequence shows a high identity (83-94%) with Hsc70s of other insects and grouped with Hsc70-3 among 5 Hsc70 members of Drosophila melanogaster. During development the grp78 transcript level was high in egg, feeding larval and adult stages but low in molting and wandering larval and pupal stages. Particularly its level was higher in the gut than integument and fat body of fifth instars. Furthermore its level was greatly decreased when fifth instar larvae were starved for 48 hrs but recovered at 3-6 hrs after re-fed diet. Our data suggests that grp78 is a member of hsc70 gene that belongs to ER and may have a role for energy metabolism at cellular level.