Tobacco whitefly-Bemisia tabaci is considered one of the most important pests in tropical and subtropical agriculture, as well as in production systems in glasshouses in temperate zones. Principle research on the identity of B. tabaci began with the recognition of more than one biotype differing in life history parameters, host plant associations, plant-related damage and insecticide resistance. Our laboratory strains of B. tabaci were identified and classified as biotype B and Q, through mtCOI PCR. Also, they were tested for their host plant preference and reaction to different insecticide. Biotype Q prefers to feed on red pepper and tomato, was less susceptible to tested insecticides, for instance acetamipirid, spinosad and thiamethoxam, than the biotype B (feed on tomato alone). There has been a report on the presence of gut bacteria in B. argentifolii (= B. tabaci biotype B) and its influence on the host insect processes. Hence, as a further pursuit, we examined our laboratory B. tabaci biotypes B and Q for their gut bacteria, whether these two biotypes are differed with each other. Gut bacterial strains isolated by standard surface sterilization method was identified through 16S rRNA gene sequence. Gut bacterial strains of B. tabaci biotypes B and Q and their close relatives retrieved from the public database (NCBI) indicated that the biotype B was less diversified only with four genera viz., Bacillus, Micrococcus, Pseudomonas and Staphylococcus, whereas the biotype Q diversified with six such as Bacillus, Janibacter, Micrococcus, Staphylococcus, Stenotrophomonas, and Streptomyces. Results of the present investigation suggesting that there may be a relationship with gut bacterial strains and susceptibility to insecticides and host plant preference of B. tabaci biotype B and Q.
Recently, Ussur brown katydid outbreaks caused a serious pest problem in areas of Yeongdong, Chungbuk. This study was performed to control the pest with environment-friendly method. Trap was made of PET plastic bottles that easily found near farmhouse. Attractant materials such as oak (Quercus acutissima) leaf, fruits (peach, apple, grapefruit and pear) sarcocarp or its juices, rice wine (makgeolli) and fish meal were directly applied into the manufactured trap and investigated for the attraction efficacy compared with the funnel trap. During one day, manufactured trap (fish trap) attracted the Ussur brown katydid more than funnel trap. The efficacy of attractant materials were as follows: peach juice (32.7 adults)> rice wine+fish meal (31.3 adults) > rice wine (27.0 adults) > pear juice (19.0 adults) > apple juice (17.2 adults) > fish meal (16.7 adults) > grapefruit juice (14.4 adults) > oak leaf (2.3 adults). The attractive efficacy of fruit juices to ussur brown katydid was more than fruit carcocarps, and the trap hangover 1m in height more than that on ground. The composition of rice wine and fish meal prolonged its efficacy when treated with disinfectant.
The toxicity of perilla-chinese Basil, Perilla frutescens whole plant-derived materials to third-instar of larva Plutella xylostella was examined using that of four insecticides and 5 constituents of P. frutescens from other research materials. The active principle of P. frutescens was identified as the sesquiterpenoids α-farnesene by spectroscopic analysis. In leaf-dipping bioassay, α-farnesene (LD50, 36.9) was 3.9 times more toxic than β-farnesene (LD50, 145.2) against P. xylostella larva, based on 48h LD50 values. This compound was less toxic than insecticides. Naturally occurring α-farnesene merit further study as potential diamond back moth control agent.
Incheon Intern'l Airport Regional Office of the National Plant Quarantine Service got the nematode separating funnel improved for the imported seedlings & tubers on 2007.
In the Bermann's funnel method, the nematode separating apparatus consist of funnel, tube and clips. The funnels used in our plant quarantine are bigger than those used in the general nematode lab. And the bigger funnels need the bigger tubes. As a result, the thick tubes and the weak clips are made uneasy for the holding and releasing of the water in funnel.
So the office changed the funnel into the better model in order to get rid of the tiresome handling for the clips. The main improving point is the replacement of clip & silicon tube with blocking apparatus in funnel bottom's end.
On the other hand, the check on nematode has been very high at the Incheon Inertn'l Airport Regional Office up to 50~250 frequency daily at season. In the consideration of the 7,800 inspection frequency annually, this improvement is expected to bring the effect of 400 working hour's reduction and the convenient work.
Pine wood nematode(PWN), Bursaphelenchus xylophilus, is a causal organism to induce pine wilt disease in many varieties of pine trees. PWN is mainly distributed in the East Asia including Japan, China, and Korea, but it was originally imported from the North America of the West. Over 70 species of Bursaphelenchus have been reported, but they are morphologically similar to each other. In Korea, only two species of Bursaphelenchus xylophilus, B. mucronatus (both Asian type and European type) have been reported however, a recent survey showed the distribution of extra species of Bursaphelenchus in dead trees. Three isolates, BSPD-1, BSPD-2, and BSPL-1, were identified as Bursaphelenchus thilandae, B. hylobianum, and B. doui, respectively, which was determined by both morphological and molecular biological characteristics. Both BSPD-1 and BSPD-2 were originally collected from Pinus densiflora in Namyangju and BSPL-1 came from Liriodendron tulipifera in Wanju. The morphology of each species were compared from the original descriptions focusing on male spicule and female tail and reproductive organ. A molecular diagnosis method, ITS-RFLP was applied to confirm morphological identification. Genomic DNA was extracted from a single individual nematode and ITS DNA was amplified by PCR. Amplified ITS was digested by 5 different restriction enzymes (Rsa I, Hae III, Msp I, Hinf I, and Alu I) and provided a discriminatory profile for different species of Bursaphelenchus. The three species, B. thilandae, B. hylobianum, and B. doui, are all unrecorded species in Korea.
The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors: dispersal stage and propagative stages. The dispersal stage is closely related to the migration to other host pines, whereas the propagative stage is coupled to the direct cause of pine wilt. To establish expressed sequence tag (EST) database of two life cycles of B. xylophilus, subtractive EST libraries were constructed using suppressed subtractive hybridization (SSH). From 3,072 and 3,840 sequences of dispersal- and propagative-specific stage cDNA libraries, 1,927 and 2,604 clusters were generated, respectively, which were annotated by BLASTx and Gene ontology (GO). A total of 1,112 (57.7%) and 1,215 (46.7%) clusters from the dispersal- and propagative-specific stage cDNA libraries respectively had the matched BLASTx hits (E≤10-2), among which 913 (47.4%) and 960 (36.9%) were classified into three categories in Gene ontology. From GO database, some respective stage-specific genes were searched and estimated the relative transcripts level according to stages using the quantitative real-time PCR.
The pine sawyer beetle, Monochamus alternatus, transmits the pinewood nematode (PWN, Bursaphelenchus xylophilus), causing the pine wilt disease (PWD), which gives rise to enormously economic as well as forest damage. However, PWN has been identified as a pathogen of PWD, it is very difficult to discriminate B. xylophilus from B. mucronatus in a short time, which are genetically and morphologically very similar. Therefore, it has been necessary to detect and eliminate PWN-infected trees in the forest area for the prevention of PWD transmission. Up to date, there is no report on biomarkers such as DNA and protein for the diagnosis of B. xylophilus. In this study, we produced a B. xylophilus monoclonal antiserum (D9-F10) from BalbC mice and screened its specificity with various proteins extracts. Western blot analysis revealed that the D9-F10 is only reactive with B. xylophilus protein extract among other tested protein extracts, indicating that the D9-F10 is specific for a B. xylophilus protein. Furthermore, two-dimensional electrophoresis showed the D9-F10 detects a very highly acidic protein, pI≒3.5. These results suggest that the D9-F10 monoclonal antibody is useful for the development of a diagnostic kit for the pine wilt disease.
We amplified D1 and D3 expansion segments of the 28S ribosomal RNA from 10 Suanguina moxae populations found in Korea. The amplification of the D1-D3 expansion segments of 28S gene of all populations tested produced a single PCR product approximately 1.03kb in size, suggesting the lack of D1-D3 expansion region size polymorphism among populations. The secondary structure model of 28S expansion segments D2 and D3 for Subanguina Moxae was predicted based on free energy minimization with comparative sequence analysis and new sequence alignment was conducted based on predicted secondary structure model. The predicted model was compared with previous predicted models of plant and animal parasite nematode. This predicted secondary structure model will provide valuable information to allocate positional sequence homology and reconstruction of reliable phylogenetic trees.
Trophic groups and functional guilds were studied of soil nematodes from the soils of two abandoned mines in Ilkwang and Gunbuk, South Korea to compare nematode communities between the heavy metal contaminated soil and the nonheavy metal contaminated soil. No obvious correlation was found between the level of heavy metal and the total number of nematodes statistically. However the overall densities of bacterial, hyphal, omnivorous and predatory nematodes from the non-heavy metal contaminated soil was higher than those from the heavy metal contaminated soil. Also the densities of c-p 2, c-p 3, c-p 4 and c-p 5 nematodes were higher from the non-heavy metal contaminated soil than those from the heavy metal contaminated soil. MI, MI 2-5 and ΣMI 2-5 were higher, but there were no significant differences.
This study was conducted to investigate occurrence and biodiversity of phytoparasitic nematodes in kiwifruit orchards in Korea. Soil samples were collected from 11 locations of Gosung, Goheung, Namhae, Bosung, Sacheon, Suncheon, Wando, Jangheung, Jeju, Jindo, Haenam from April to August, 2008.
Phytoparasitic nematodes were found in 115 among 178 soil samples.
The major genera of phytoparasitic nematodes on kiwifruit were Meloidogyne spp.(52.2%), Tylenchinae (42.1%), Tylenchus spp. (9.0%), Helicotylenchus spp. (9.0%) and Tylenchorhynchus spp. (6.7%). Ditylenchus spp., Hirschmaniella spp., Trichodorus spp. and Psilenchus spp. were also detected, even though their frequencies were very low. According to the cultivation period, the phytoparasitic nematodes were found as follows; 16.9% of soils under 5 years of kiwifruit cultivation, 10.7% 6~10 years, 23.0% 11~15 years, 3.9% 16~20 years and 5.6% over 21 years respectively. The frequency of phytoparasitic nematodes is higher in the open field (50.0%) than that in plastic houses (13.5%). The frequency of phytoparasitic nematodes was 36.5% in the field where vegetables were previously cultivated and 12.9% in the field where rice was previously cultivated.
Metamorphosis is a development process involving the programmed cell death of obsolete larval organs. Aspartic proteinase cathepsin D (BmCatD) is involved in the silkworm Bombyx mori metamorphosis. Here we show a novel functional role of cysteine proteinase cathepsin B during B. mori metamorphosis. The B. mori cathepsin B (BmCatB) was expressed in the fat body, epidermis, ovary, testis, and hemocyte of the larval and pupal stages. The BmCatB was ecdysoneinduced, expressed in the fat body of the molting, the final larval instar and pupal stages, and its expression led to programmed cell death. RNA interference (RNAi)-mediated BmCatB knock-down inhibited the programmed cell death of larval and pupal fat body, resulting in the arrest of larval-pupal transformation. BmCatB RNAi is up-regulated the expression of BmCatD. Based on these results we concluded that BmCatB is critically involved in the histolysis of the larval and pupal fat body, indicating that BmCatB and BmCatD are mutally regulated during silkworm metamorphosis.
Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2,340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1,393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5’UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.
Mortality of honeybees is a serious problem that beekeepers have to face periodically in Korea and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Deformed wing virus(DWV), Israle Acute Paralysis Virus (IAPV), Black queen cell virus (BQCV), Cloudy wing virus(CWV), Kashmir bee virus(KBV), Sacbrood virus(SBV), Chronic bee paralysis virus(CBPV) in samples of korea honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in all provinces, simultaneous co-infection of colonies by several viruses and the fact that 96.3% of the samples were infected with one or more virus, indicates they are widely spread in the region. Using uniplex and multiplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including DWV, IAPV, BQCV, KBV, CWV, and described the detection of mixed virus infections in bees from these colonies. Conclusively, investigated disease of the bee, and confirmed new virus that lead to bee disease, this is thought by valuable thing as data for development of beekeeping industry such as CCD(Colony Collapse Disorder)'s cause searching examination.
Viruses of the honeybee, Apis mellifera L. are known to reside at low levels in colonies, typically showing no apparent signs of infection. Chronic paralysis virus(CBPV) is known to induce significant losses in honey bee colonies. The pathology is characterized by clusters of trembling, flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive entrance. A minusstrand-specific RT-PCR was used to assess viral replication. This is the first report on the infection of CBPV in Korea. Using (-)RT-PCR, 27 apiaries in korea were screened for the honeybee viruses, with positive colonies being analysed for viral genetic diversity. We got 550-nt PCR product from CBPV genomic RNA. Nucleotide sequences were aligned to the complete CBPV genomic RNA sequence deposited in the GenBank database and was revealed 96%(AM-CBPV) identity, respectively. Sequence comparison with other CBPV and honeybee virus.
Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.
Acetaminophen (CAS 103-90-2) is one of the most used pharmaceuticals around the world. In Korea, it was produced 1,069 tons in 2003. This chemical is not eliminated in wastewater treatment plant and may flow into the ecosystem through various routes. Therefore, there is a possibility that it can make an adverse effect on aquatic organisms. To examine its ecological toxicity, we used three native Korean aquatic invertebrate species, Daphnia sp., Chironomus yoshimatsui, and Ephemera orientalis. The acute toxicity on Daphnia sp. was moderately high, and its 48 hour median immobilization concentration (EC50-immobilization) was 51.7 mg/L. On the other side, the reproductive toxicity was very high, and its EC50 of 25 day reproduction test was 0.005 mg/L. In E. orientalis egg hatching test, the median egg hatching inhibition concentration was 0.199 mg/L. C. yoshimatsui was most tolerant to acetaminophen, in which 48 hour median lethal concentration (LC50) was 400.0 mg/L and 45 day median emergence inhibition concentration (EC50-emergence) was 45.27 mg/L. From this results, we concluded that acetaminophen is hazardous to freshwater macroinvertebrates, especially to water flea. Therefore we need to study more about pharmaceuticals' ecotoxicology including acetaminophen and to assess their potential ecological risk.
The nucleotide sequence of the Spodoptera litura granulovirus (SlGV) genome was determined and analysed. It was 124,121 bp long, with a 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 nucleotides or larger. The 133 putative ORFs covered 86.3% of the genome. Among these, 29 ORFs were conserved in most completely sequenced baculovirus genomes, 44 were granuloviruses (GVs)-specific, 4 were nucleopolyhedroviruses (NPVs)-specific, and 56 were present in some NPVs and/or GVs. Especially, we proved that there were 9 SlGV-specific ORFs in 44 GV-specific ORFs by RT-PCR. Chitinase and cathepsin genes involved in the liquefaction of the infected hostwere not found in the SlGV genome, which explains why SlGV-infected insects do not degrade in a typical manner. When the phylogenic relationship was analyzed using the nucleotide sequence of granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which were belonged to TypeI granulovirus.
To find some antibacterial peptides responsible for bacterial resistance, we performed differential hybridization with total cDNA probes which synthesized from normal and immunized larvae. Thirteen individual cDNA transcripts were expressed differentially in a total 1,862 random cDNA clones. One of upregulated genes is a novel member of the insect defensin-like peptide(Coprisin), a family of antibacterial peptide. Northern blot analysis showed that Coprisin was up-regulated at 4h and reached the highest point level at 16h after injection of E.coli. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with predicted molecular weight of 8.6 kDa and PI of 8.72. Comparison of the deduced amino acid mature portion of Coprisin with defensin-like peptide of other insect indicated that it has 79.1% and 67.4% identity with Anomala cuprea and Allomyrina dichotoma, respectively. To find antibacterial active region of Coprisin, we synthesized four peptides corresponding to amino acid residues 1V-43N-NH2(CopN1), 5-16(CopN2), 19-30(CopN3) and 31-43(CopN4) of coprisin having amidated amino acid residues at their Cterminal. A 12-mer amidated at its C-terminus, ACALHCIALRKK-NH2 (Ala19-Lys30-NH2) was synthesized based on the deduced amino acid sequence, assumed to be an active site sequence. This peptides showed antibacterial activity against E.coli, Staphylococcus aureus, MRSA, Psedomonas syringae, and Pectobacterium carotovorium. Modified 9-mer peptide, LRCIALRKK-NH2, showed strong antibacterial activity than mellitin peptide used as a positive control against gram-negative and gram-positive bacteria. This peptide showed no haemolytic activity and quite stable at 100℃ for several hours of incubation and in a wide pH range(pH2-12). Therefore, this peptide may be a good candidate for the development of new drug with potent antibacterial activity without cytotoxicity.
The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.
To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.