,  , The larvicidal activities of Piper nigrum fruit methanol extracts and its constituents against larvae of the diamondback moth, Plutella xylostella, were investigated using the leaf dipping method. Administering the P. nigrum methanol extract resulted in 100 and 97% mortality against diamondback moth larvae at 5.0 and 2.5 mg/mL concentrations, respectively. Larvicidal activities of the P. nigrum fruit-derived piperidine alkaloids, piperine, and N-isobutylamide, as well as pellitorine, guineensine, pipercide, and retrofractamide A against P. xylostella varied according to test compound. Based on the 48 h LC50 values, the most toxic compound to diamondback moth larvae was guineensine (0.013 mg/mL) followed by retrofractamide A (0.020mg/mL), pipercide (0.033mg/mL), and pellitorine (0.046 mg/mL). The LC50 value of piperine was >, 0.5 mg/mL.

From the methanol crude extracts of the tree of heaven (Ailanthus altissima) leaves, the antifeedant substance was isolated and bioassayed with different concentrations against diamondback moth (Plutella xylostella) larvae. The antifeeding activity was evaluated by measuring the feeding area during 24 hr after inoculation. Methanol extracts showing antifeeding activity at 5000 ppm was subsequently fractionated into hexane, chloroform, ethyl acetate and water layer. Third larvae of diamondback moth was tested to each fraction layer. Chloroform layer shows the highest antifeeding activity and the layer was purified by silica gel open column chromatography. The C22 and C23 fractions showed higher antifeeding ratio with 96 and 86%, respectively, and then these two fractions were re-isolated by ODS open column chromatography. As a result, both fractions in methanol 40% (v/v) showed antifeeding ratio over 90%. The C221 fraction showed insecticidal activity in all fraction, however, C231 fraction was showed the antifeeding activity only in C2311 fraction. The C2311 fraction judging to have antifeeding activity was re-isolated and purified by HPLC and recycling, and finally obtained the bioactive substances (C23111) with antifeeding ratio with 88%. The structure of bioactive materials isolated was confirmed by LC-mass and 1H-NMR(500 MHz), 13C-NMR(100 MHz).

This study was performed to investigate the antifeeding activity of Perilla frutescens extracts against diamondback moth, Plutella xylostella larvae and to confirm the electrophysiological responses of two sensilla (LST=lateral styloconic sensillum, MST=medial styloconic sensillum) in maxilla galea, a chemoreceptor. Crude extract of P. frutescens in methanol was showed antifeeding activity approximately 70%, and subsequently separated into four fractions - n-hexane(H), chloroform(C), ethylacetate(E), and water(W). Antifeeding activity was only showed in n-Hexane fraction around 99%. H1, H2, H3, H4, H5 were isolated from n-Hexane fraction using an open column chromatography, and the bioassay showed strongest antifeeding activity in H1 fraction. Using an oscilloscope, electrophysiological responses of two sensilla showed more seven times activity in MST. H11, H12, H13, H14 were separated from H1 fraction and antifeeding activity was showed highest in H11 fraction. H11 fraction was examined electrophysiological responses at doses of 100, 10, and 1 ppm, and MST of P. xylostella responded at a dose of 100 ppm. H11 fraction was separated using HPLC and identified using GC/MS and NMR. Finally, the structure of active compound proved to be farnesene with molecular weight of 204, and a formula of C15H24

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth (Plutella xylostella) type-1 acetylcholinesterase locus, putatively involved with organophosphate and carbamate insecticideresistance. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generatedfrom the plots by linear regression, and the signal ratios were shown to highly correlated with resistance allele frequencies (r2>0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations. As judged by inhibition assay, all populations showed resistance to paraoxon, DDVP, carbaryl, and carbofuran. In addition, different DBM strains exhibited differential sensitivities to both OPs and CBs depending on the structure of inhibitor, implying that the resistance of DBM against OPs and CBs is saturated and widespread in Korea.

The diamondback moth, Plutella xylostella, is one of the most important pests of cole crops in the world and is the first insect to evolve resistance to Bt toxins in open-field populations. To search for useful molecular markers for Bt reistance monitoring, the PCR-based restriction fragment length polymorphism (RFLP) profiles of three aminopeptidase N (PxAPN1, PxAPN2 and PxAPN4) were determined for 15 representative regional field populations of P. xylostella. Most regional samples had similar RFLP patterns, whereas PxAPN1 from four regions and PxAPN4 from two regions showed different banding patterns after restriction enzyme treatment, but no differences were found in PxAPN2 among populations. The DNA sequence analysis revealed that a point mutation at the restriction site was responsible for the polymorphism of PxAPN1 but no mutations were observed in PxAPN4. Comparing amino acid sequences of PxAPNs from regional populations with reference PxAPNs (GenBank accession no. AAB70755) revealed that four regional populations possessed a point mutation in the Cry1A binding site of PxAPN1 and five regional populations possessed a deletion of eight amino acids in PxAPN4. These RFLP patterns were consistently observed in Southern regions of Korea, including Kyungsangnam-Do and Jeju-Do. The functional association of these RFLP with Bt resistance is currently under investigation

Diamondback moth (DBM, Plutella xylostella L.), the most destructive pest of cruciferous crops, is well-known as typical subtropical insect pest. A number of biological agents such as diseases, parasites and predators can affect populations of DBM in the fields negatively. In previous reports, we suggested Cotesia glomerata, Diadegma semiclusum, and Microplitis plutellae as promising natural enemies to DBM control at highland areas, but these species are larval parasitoids. In 2004~2009, we searched highland fields cultivating various cruciferous crops for PUPAL parasitoids which can supplement the unsatisfactory parasitism in the augmentative release of larval parasitoids. We obtained adults of hymenopteran parasitoid from DBM pupae in early July at Hoengseong region (asl 540 m), and then identified as Diadromus sp., although being a critical species so far. This parasitoid showed high rate of parasitism, about 13.2%, in mid October at the same region. Development period from oviposition to emergence of Diadromus sp. ranged from 14 to 18 days under 23℃ condition. Adult longevity, > three weeks, was longer when it was provided with 10% sugar solution as food than with water only or without food. Male adults mated as soon as emerging from parasitized DBM pupae, and laid eggs into DBM pupae for a week. Parasitism by Diadromus sp. was highest on 7th day after emergence. In contrast, lethality of DBM pupae which were not parasitized by wasps showed 60% on average for seven days. Parasitized DBM pupae could be stored at 10℃ for two weeks. The simultaneous augmentative release of larval parasitoids and pupal parasitoids can be an important component to integrated DBM management program in the future.

The life table statistics of diamondback moth on the transgenic Chinese cabbage (line: SKCP) with myrosinase gene was compared with that on the non transgenic Chinese cabbage (line: SC) at 25±1℃. Adult life span and number of progeny of P. xylostella on the SC and SKCP lines were similar to each other. The sex ratio, developmental period, intrinsic rate of increase and finite rate of increase of P. xylostella on SC and SKCP were not significantly different in both treatments (sex rati t=-1.60; df=220; P=0.1108, developmental period: t=-0.55; df=220; P=0.5803, intrinsic rate of increase: t=-0.11; df=45; P=0.9172). However, the finite rate of increase and net reproduction were significantly different in between SC and SKCP lines (finite rate of increase: t=2.26; df=45; P=0.0287, net reproduction: t=2.08; df=45; P=0.0442). This work was supported financially by Biogreen21 project of Rural Development Administration (No. 20070301-034-010)

Bacillus thuringiensis (Bt) is effective to control the diamondback moth, Plutella xylostella. However, its relative slow and unstable control efficacy limits its wide use by farmers. To facilitate pathogenic rate of Bt, a bacterial mixture technique has been developed in this study. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), possess high immunosuppressive activity against several lepidopteran insects. The mixture treatments using Bt + Xn or Bt + Ptt significantly enhanced Bt pathogenicity in median lethal concentration and time. Though live Xn and Ptt bacterial cells gave significant effect on the pathogenicity, their 48 h culture broth after removing the bacterialcells still possessed the synergistic effect on the Bt pathogenicity. The larvae fed with the bacterial culture broth suffered significant immunosuppression in response bacterial to infection

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp encoding two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28~50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. ORF301 and ORF302 were transiently expressed in hemocyte, fat body, gut, and epidermis of P. xylostella. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to non parasitized larvae of P. xylostella, in which the two genes were expressed at least for four days post-injection. The P. xylostella larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population, suppression of immune associated genes including cecropin, pro-phenoloxidase (PO) and serpin1, and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression by the viral segmenttreated larvae, but not by ORF302 specific dsRNA. The larval injected with CpBV-S3 exhibited an enhanced susceptibility to baculovirus infection. These results indicate that ORF301 of CpBV-S3, which containing BEN domain, suppresses both cellular and humoral immune responses in P. xylostella.

Diamondback moth (Plutella xylostella L.) belonging to genus Lepidoptera is a notorious pest of cruciferous crops worldwide. We evaluated the bioinsecticidal activity of the liquid cultures (LB and NB) of a bacterial strain, Serratia sp. EML-SE1, isolated from a diseased diamondback moth. The pathogenicity of a bacterial strain to diamondback moth was confirmed by the following procedures: treatment of liquid culture on cabbage leaves, ingestion of inoculated cabbage and mortality response. For the test, twenty 3rd instar larvae of diamondback moth were placed on the Chinese cabbage leaf in a round plastic cage (Ø 10 × 6 cm) and sprayed with the liquid cultures. After 72 hours, insecticidal activity of LB and NB cultures of Serratia sp. against P. xylostella larvae showed 91.7% and 88.3%, respectively. In addition, the bioinsecticidal activity on potted cabbage with 14 leaves in a growth cage (165 × 83 × 124 cm) also was similar to that of plastic cage experiment. Summarized, the Serratia sp. EML-SE1 may be a potent candidate as a bioinsecticidal agent to control diamondbac kmoth.

Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.

This study was conducted to develop the economic threshold for the diamondback moth (Plutella xylostella (L.) on Leaf Broccoli (leaf vegetable) in 2007. To investigate the relationship between initial density of diamondback moth larvae and broccoli leaf yield, experimental plots with five treatments (0, 0.5, 1.0, 2.0, 4.0 larvae per plant) as initial density were established. We inoculated larvae in chesses cloth covering to survey larvae density change. When grown to eat, leaves of broccoli were harvested by periods. 60-70% of larvae were removed whenever we picked the leaves. High levels of larvae were associated with significant reductions in leaf yield. There were 85%, 64%, 58%, and 56% yield reductions from the diamond back moth larvae density in 0, 0.5, 1,0, 1.5, 2.0 per plant, respectively 25days after larvae inoculation. The regression equation used to predict leaf yield based on the number of initial larvae density per a plant was y=1635-393x(R2=0.79***). The economic injury level of diamondback moth on leaf broccoli was 2-3 larvae per 10 plants for a damage level of 5%. The economic thresholds was 1-2 larvae per 10 plants. Thus, the diamond back moth management should be initiated 1-2 larvae occurrence per plant.

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral and antimicrobial responses. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

Hemolin is a member of the immunoglobulin (Ig) superfamily and contains four Ig domainsthat are similar to neural cell adhesion molecules. It has been regarded as a recognition molecule at immune challenge in insects. This study showed that hemolin of Plutella xylostella was expressed during pupal and adult stages but absent in all larval instars without any immune challenge. It is, however, strongly induced by the injection of Escherichia coli or its lipopolysaccharide in hemocytes, fat body and gut. A double-stranded RNA (dsRNA) interference experiment revealedits role in activation of prophenoloxidase (PPO) in the hemolymph during bacterial infection. Also its involvement in cellular defense was investigated in its mediation of the adherence of hemocytes to rat blood erythrocytes which was knocked down by its dsRNA. Finally, its physiological significance in pupal stage was confirmed by using dsRNA, which significantly prevented adult development. Therefore, it is concluded that hemolin plays roles in both immune and adult development in P. xylostella.

The diamondback moth, Plutella xylostella, is reluctant to a baculovirus, Autographa california nucleopolyhedrosis virus (AcNPV) at its oral administration. However, parasitization by an endoparasitoid wasp, Cotesia plutellae, enhances the viral susceptibility. This study analyzed an antiviral activity of P. xylostella in response to the viral infection and determined the parasitic factor inhibiting the antiviral mechanism. For the analysis of antiviral activity of P. xylostella, a recombinant AcNPV expressing enhanced green fluorescence (AcNPV-EGFP) was orally adminstered to lavae of P. xylostella. After 24 h, EGFP expression was observed in the midgut tissue at a confocal-FITC mode. At the same time, a characteristic midgut melanotic response (MMR) was observed in some midgut regions under a phase contrast microscope. Thereafter, the EGFP signal was attenuated, while MMR spread on most midgut region. When the MMR was scored from 0 to 5 by the intensity of melanized cell density, it increased in time- and dose-dependent manners at the viral administration per os. These results suggest that the MMR is an antiviral activity of P. xylostella. This antiviral activity was significantly attenuated by C. plutellae parasitism. The parasitized P. xylostella showed significant decrease in the MMR score compared to nonparasitized larvae when they were orally administered with the same dose of AcNPV. To determine the parasitic factor(s) inhibiting the antiviral activity from the symbiotic polydnavirus of C. plutellae (C. plutellae bracovirus: CpBV), CpBV-IkB, which is a viral homolog of NFkB inhibitor and has been considered as an antiviral factor as in other polydnaviruses, was tested. A recombinant AcNPV expressing CpBV-IkB (AcNPV-IkB) was constructed and administered to P. xylostella larvae. As expected, AcNPV-IkB significantly decreased the antiviral activity measured by the MMR score compared to AcNPV-EGFP treatment. This study suggests that CpBV-IkB plays an antiviral parasitic role in the molecular interactions between P. xylostella and C. plutellae.

Many female moths produce and release sex pheromones to mate successfully with a conspecific male. Sex pheromone production in lepidopteran moth is known to be under the regulation of a pheromone biosynthesis activating neuropeptide (PBAN). A PBAN polypeptide is processed into five neuropeptides (NPs) after post-translational modification, resulting in diapause-like NP, PBAN, α-, β- and γ-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure. PBAN (Plx-PBAN) from Plutella xylostella consists of 30 amino acids, the shortest PBAN so far reported. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1 h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in both sexes, with the highest expression level in the head of female adults. Plx-PBAN binds to its receptor on pheromone gland cells. PBAN receptor has seven transmembranes, indicating G-protein coupled receptor family and transduces its signal via G-protein mediated signal transduction. Subsequently, calcium channels remain activated and stimulate biochemical reactions for sex pheromone production in the pheromone gland.

Five strains of Korean entomopathogenic nematodes (EPN), steinernematids and heter-orhabditids were evaluated and tried in laboratory, pot, and vegetable greenhouses for environmentally friendly control of diamondback moth (DBM), Plutella xylostella, from 2002 to 2005. LC₅₀ values of five EPN strains against DBM were different depending on nematode strain and DBM instar. LC₅₀ value of Steinernema carpocapsae GSN1 (GSN1) was the lowest representing 2.6~3.9 infective juveniles (Ijs, 3rd stage) to 2nd to 4th instars of DBM. Pathogenicity of five effective strains against DBM was different depending on nematode strain, concentration, application times, and vegetable in pot. The most effective nematode was GSN1. Steinernema spp. was more effective than Heterorhabditis spp. against DBM. Two or three times of applications of EPN were effective regardless of nematode strain and concentration in pot. Efficacy of EPN was different depending on vegetable species. Efficacy was higher on Chinese cabbage, red mustard, and Ssamchoo than that on cabbage, kale, and leaf broccoli. Efficacy of GSN1, Steinernema GSNUS-10, Steinernema GSNUS-14, and Heterorhabditis GSNUH-l was variable depending on nematode strain, concentration, application times, and vegetable in greenhouse experiments. GSN1 was the most effective and 100,000 infective juveniles per ㎡ (=1 × 10⁹ Ijs/ha) resulted in higher efficacy. Three times of application of nematodes led to higher control efficacy than one or two applications. Efficacy of nematodes was higher on Chinese cabbage than cabbage or kale in greenhouse.

Inhibitor <SUB>K</SUB>B (I<SUB>K</SUB>B)-like gene has been found ill the genome of Cotesia plutellae bracovirus (CpBV), which is the obligatory symbiont of an endoparsitoid wasp, C. plutellae. The open reading frame of CpBV-I<SUB>K</SUB>B was 417 bp and encoded 138 amino acids. Four ankyrin repeat domains were found in CpBV-I<SUB>K</SUB>B, which shared high homology with other known polydnavirus I<SUB>K</SUB>Bs. Considering a presumptive cellular I<SUB>K</SUB>B based on Drosophila Cactus, CpBV-I<SUB>K</SUB>B exhibited a truncated structure with deletion of signal-receiving domains, which suggested its irreversible inhibitory role in NF<SUB>K</SUB>B signal transduction pathway of the parasitized host in response to the wasp parasitization. CpBV-I<SUB>K</SUB>B was expressed only in the parasitized diamondback moth, Plutella xylostella. Its expression was estimated by quantitative RT-PCR during parasitization period, showing a constitutive expression pattern from the first day of parasitization. An indirect functional analysis of CpBV-I<SUB>K</SUB>B was conducted and suggested a hypothesis of host antivirus inhibition.