The nematicidal activity of Phellodendron amurense rhizome-derived materials (methanol extract) toward Meloidogyne Spp. second-stage juveniles (J2) and these effects on Cucumis sativus and Cucumis melo. Results were compared with these of fosthiazate. J2 was examined using 24-well plate tests, pot bioassays (C. sativa and C. melo) and Field trials (C. melo). In 24-well plate test with J2, methanol extract of P. amurense exhibited 98.7% and 69.8% mortality at 0.25 and 0.125 mg/ml toward J2, respectively, whereas Fosthiazate showed 100% mortality at 1 mg/ml. In pot bioassays with J2, P. amurense rhizome methanol extract gave 79.5% and 57.4% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. sativa and 73.7% and 53.3% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. melo, respectively. In Field test at C. melo in greenhouse showed 55.1% and 26.9% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x) applied soil. P. amurense rhizome-derived materials, merit further study as potential root-knot nematode control agents because of their nematicidal activity.

Injection of nematicides such as emamectin benzonate and milbemectin is the most common practice to control the pinewood nematode, Bursaphelenchus xylophilus, in Korea. These macrocyclolactone nematicides, however, are expensive, limiting their practicability despite of high efficacy. In an attempt to screen affordable alternative organophosphate (OP) and carbamate (CB) nematicides, we identified and characterized three acetylcholinesterases (ACE, EC 3.1.1.7) from B. xylophilus and functionally expressed them using baculovirus system. In inhibition assay using 11 OPs and 3 CBs, all the three ACEs were highly inhibited by paraoxon, DDVP, chlorpyrifos-oxon and mevinphos of OPs and carbofuran and carbaryl of CBs but not inhibited well by the others. Interestingly, inhibition assay revealed that BxACE-3 is less sensitive to all insecticides tested than other two ACEs. In additional bioassay, chlorpyrifos, DDVP and parathion showed a high LC50 but all CBs tested did a very low mortality. The inhibition kinetic data and bioassay data obtained in this study should provide essential information for the development of OP-based nematicidal agents against B. xylophilus. Availability of expressed ACE will also facilitate the development of in vitro screening system to develop potential OP nematicides.

The ovarian development and oviposition response of two vector beetles of pine wood nematode, Monochamus alternatus and M. saltuarius, was investigated. The ovary structure of two beetles was investigated by means of light microscopy. The ovary of both beetles was composed of two ovaries that are connected by a common oviduct. Each of the two ovaries was consisted of twelve ovarioles, the functional units of female oogenesis. The ovary type was meroistic telotrophic with nurse cells and oocytes in the tropharium. Ovarian development of two vector beetles was completed at 12-14 days after maturation-feeding pine twigs. Aspect of accumulation of vitellogenins in the ovary of two vectors showed difference in developmental stages and major yolk proteins, differently from vitellogenin. To investigate oviposition performance of two vector beetles, we provided P. thunbergii and P. koraiensis bolts for egg laying and collected emerged adults from each pine bolts in the next year. M. saltuarius females made more oviposition wounds and entrance hole of larvae than M. alternatus on pine bolts. We also investigated whether two beetles can transmission of Bursaphelenchus xylophilus and the European type of B. mucronatus via oviposition to noninfected dead pines. 48-83% from newly emerged adults of two beetles were carried two species nematode. This results suggested that two species beetles can transmit nematodes through oviposition performance and transmitted nematodes successfully propagate in non-infected dead pines.

Pine wood nematode (PWN), Bursaphelenchus xylophilus is associated with the pine wilt disease and transmitted by pine sawyer, Monochamus alternatus. Because pine sawyer has one-year life cycle, one natural infection of PWN is occurred a year. Therefore, artificial propagation method of PWN is needed to improve experiment associated with PWN. In this study, effect of diameter, paraffin sealing of twig and dosage on pine wood nematode reproduction in Japanese black pine, Pinus thunbergii. PWN reproduction was compared in twigs of P. thunbergii and P. densiflora. Numbers of reproduced PWN were higher with decreasing diameter of twig. Distance (5 and 10 cm) from inoculation site of PWN did not influence reproduction of PWN. Reproduced numbers of PWN were higher in the paraffin-sealing twig than non-sealing twig. Dosage of PWN influenced reproduction of PWN. Reproduction rate was the highest at the rate of 10 IJs (13.7 and 61.1-fold increasing in P. densiflora and P. thunbergii, respectively 30 days later) whereas lowest at the rate of 1000 Ijs (1.1 and 0.7-fold increasing in P. densiflora and P. thunbergii, respectively 30 days later). Numbers of reproduced PWN were more in P. thunbergii than P. densiflora.

Venom allergen-like protein 2 (Vap2) was characterized from the pinewood nematode, Bursaphelenchus xylophilus, which is a destructive pathogen in several countries including Japan, China and Korea. Among three vaps of B. xylophilus (Bx-vap)reported in GenBank, Bx-vap2 showed the highest transcript level in both pine-grown propagative stage (PGPS) and media-grown propagative stage (MGPS). Bx-vap2 also was revealed that its transcript level over 10-fold increased in PGPS. In addition, western blot using BxVap2-polyclonal antibody showed that expression level of BxVap2 was significantly increased in PGPS. In immunohistochemistry, moreover, strong signals were detected around putative subventral gland in PGPS, whereas weak signals were observed in MGPS. These experimental results suppose pathogenic function of BxVap2 and migration assay using Bx-vap2 knock-down worms by RNA interference supports this postulation.

In this study, we investigated the nematicidal activities of 102 Korean domestic plant extracts which have not been tested before against B. xylophilus. Nematicidal activity of plant extracts were varied according to plant species and extraction part. Among 102 plant extracts, 42 plant extracts in 22 families showed >50% nematicidal activity against pine wood nematode at 10 mg/mL concentration. Among 31 wood extracts, very strong nematicidal activity (100%) was produced from extract of Neolitsea aciculate, Prunus sargentii and Rhus chinensis at 10 mg/mL concentration. Extracts from Camellia japonica, Carpinus cordata, Daphniphyllum macropodum, Eucommia ulmoides, Ilex cornuta, Myrica rubra, Vaccinium bracteatum, and Zelkova serrata showed strong nematicidal activities (80-99%). The other samples exhibited activities <80%. In a test with leaf extracts, strong neamticidal activity was observed in extract of Machius japonica (100%) followed by Picea koraiensis (99.4%), Meliosma oldhamii (82.7%), and Zelkova serrata (81%). The other 37 plant species revealed <80% mortality. Strong nematicidal activity (>80%) was observed from bark extract of Carpinus cordata, Comus kousa, Cryptomeria japonica, Machius japonica, Quercus glauca and Zelkova serrata. Nematicidal activity of other 19 bark extracts was less than 80%. Twig extract of Smilax china and fruit extract of Forsythia koreana showed 86.6 and 81.4% nematicidal activity at 10 mg/mL concentration, respectively.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) is known to be a major pathogen of the pine wilt disease (PWD). However molecular pathology of B. xylophilus is not completely understood, the pathogenecity of PWD is related to cell wall-degrading enzymes such as endoglucanases, expansins and pectate lyases (PELs). Recently, we developed stage-specific expressed tag library of B. xylophilus and identified a novel PEL, Bx-PEL3. We cloned Bx-PEL3 gene with RT-PCR, which showed high similarity to previously reported Bx-PELs. Phylogenetic analysis revealed that PEL3 was much closer to PELs of B. xylophilus than any other PELs. PEL3 has a conserved intron site as found in Bx-PEL2 in the genomic DNA analysis. Quantitative real-time PCR analysis revealed that Bx-PEL1 and Bx-PEL2 were more predominantly expressed than the Bx-PEL3 in B. xylophilus. The difference of expression level among Bx-PELs according to growth condition suggests that each Bx-PEL plays different biochemical role in the pathogenesis of the PWD.

The oral toxicities of symbiotic bacteria Photorhabdus temperata ssp temperata (Ptt), mutually associated with entomopathogenic nematode Heterorhabditis megidis, and P. luminescens ssp. laumondii (TT01) with H. bacteriophora, were demonstrated to adults of the sweetpotato whitefly Bemisia tabaci. Sucrose solution (25%) containing bacteria-free supernatant of culture media of symbiotic bacteria was ingested into adult whiteflies within the glass tube. Whitefly mortalities were shown similar patterns against two bacterial media. Mortalities were significantly increased to 60-64% at 36 hours and almost 100% at 60 hours after treatments. In addition, We demonstrated the effect of oral ingestion of symbiont culture media on the gene expression of B. tabaci. Several genes fluctuated those expression levels. Our results suggest that oral ingestion of symbiont culture media of entomopathogenic nematodes significantly changed metabolic rates and highly lethal to whiteflies. The use of symbiotic bacteria of entomopathogenic nematodes provides a great potential as an alternative genetic resource of Bacillus thuringiensis, a major resource of microbial insecticide.

It is well known that the root-knot nematodes, Meloidogyne spp., incite and aggravate the diseases caused by fungal and bacterial pathogens. The synergistic effects of the inoculation of Meloidogyne incognita combined with Fusarium oxysporum f. sp. lycopersici showed the greatly increased wilt symptoms developed on tomato plants compared to the inoculation of either of the two pests alone. For the biological control of the complex disease, a variety of bacterial isolates were tested for antagonistic effects to select ones that had both nematicidal and antifungal activities. Among forty plant growth-promoting rhizobacteria (PGPR) tested, Paenibacillus polymyxa G508 and G462 and P. lentimorbus G158 showed strong antifungal and nematicidal activities against F. oxysporum f. sp. lycopersici and second-stage juveniles (J2) of M. incognita, respectively, and also inhibited egg hatch of the nematode. The addition of Paenibacillus strains into potted soil suppressed the Fusarium-wilt severity and root galling on tomato and increased plant growths. P. lentimorbus G158 were abundantly proliferated on tomato seeds and hypocotyls more than P. polymyxa G 462 and had no phytotoxic effect on tomato plant. Under the greenhouse conditions, seed treatment of P. lentimorbus G158 reduced wilt severity caused by Fusarium wilt-root knot disease complex and root gall formation and increased tomato growth compared to the untreated control. Root-galls caused by both pathogens treated with bacterial culture had fewer and smaller giant-cells than untreated control, and scanning electron microscopy revealed alteration and distortion of hyphal cell wall of F. oxysporum and lysis of M. incognita egg shell by the bacterial treatment. All of these results suggest the Paenibacillus strains, especially G158 may have a high potential developed as biological control agents for the complex disease.

작물육종 프로그램에 선충저항성은 매우 중요한 구성요소의 하나이다. 세계적으로 선충에 의한 작물의 수량 감소는 약 10% 정도로 추산되고 있으나 아시아 각국에서는 주요 작물별로 선충의 피해현황이나 저항성 작물 등에 대한 연구가 아직 미진한 실정이다. 본 연구에서는 한국, 베트남, 말레이시아 각국의 최근 선충저항성 관련 연구 현황을 분석하였다. 한국에서는 주요 경제작물 중 쌀, 콩, 고추, 오이, 참외 뿐만 아니라 장미, 카네이션, 국화 등 화훼작물에 대한 선충저항성 품종 선발 연구가 이루어졌다. 베트남에서 는 바나나, 커피, 쌀, 리치 등에 대한 선충저항성 검정연구와 식물 기생선충의 분포에 관한 연구가 이루어졌다. 말레이시아에서는 최근 파인애플에서 선충으로 인한 연작장해가 문제가 되고 있으나 저항성품종 개발 연구는 시작단계에 있다. 아시아 각국에서 선충에 대한 연구가 부진한 이유는 방제전략의 부재, 경제적인 종합방제 대책의 부족, 선충에 대한 정보 부족, 살선충제 가격 부담 등이 들 수 있다. 따라서 보다 경제적이면서 지속가능한 선충관리전략의 수립이 시급하다. 선충저항성 유전자원의 이용과 품종개발은 아시아 지역에서 경제적이고 효과적인 선충관리 대책의 하나가 될 수 있다.

This study was conducted to evaluate the effects of temperature and nematicides on endospore attachment characteristics of 2 Korean isolates of nematodeparasitic bacteria, Pasteuria penetrans. P. penetrans CJ-1 and 98-35 isolates and 3 species of root-knot nematodes in U. of Florida were used ; Meloidogyne incognita (MI), M. arenaria (MA), and M. javanica (MJ). Three nematicide levels of 2, 4, and 6 ppm were compared by treating Vydate and Nemacur in the endospore containing soils. The endospores were attached to the root-knot nematode juveniles by centrifugal method. Reproduction of the isolates was checked by observing root-knot nematode females harvested from inoculated tomato roots. 1. Among the two Korean isolates, 98-35 showed attachments to MI, MA, and MJ juveniles, but it could not reproduce in any species. CJ-1 reproduced on all the 3 tested root-knot nematode species. 2. Endospores pretreated in -14°C showed higher attachment rate than those pretreated in 4, 24, and 40℃. 3. Nemacur treatments significantly suppressed the attachment of the endospores to the juveniles, while Vydate treatment effects were negligible.

tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.

Effect of inoculation level on pathogenicity, development, and propagation of entomopathogenic nematode, Steinernema arenarium was investigated using the last instar of great wax moth, Galleria mellonella. Pathogenicity of S. arenarium was higher with increasing inoculation level representing 82% at the rate of 5 infective juveniles (IJs) while >98% at the rate of >10 IJs. The number of IJs penetrated into the host was 2.7, 5.0, 7.4, and 12.2 at the rate of 5, 10, 20, and 40 IJs, respectively while 24.3 at the rate of 80 IJs and 40.2 at the rate of 160 IJs. Inoculation level did not affect female adult size (4,616 to 6,444 ㎛) while affected male adult size (1,600 to 1,934 ㎛). The rate of stunted female adults was 70.2% at the inoculation level of 80 IJs and 63.7% at the inoculation level of 160 IJs. The number of progenies was 20,431, 26,696, 47,943, 50,516, 58,701, and 74,235 at the rate of 5, 10, 20, 40, 80, and 160 IJs, respectively. The body lengths of IJs were different depending on inoculation level ranging from 636 to 1,496 ㎛.

This study was conducted to identify plant-parasitic nematodes and determine the distribution pattern of the nematode species in citrus fruit orchards in Korea from April to August, 2008. Plant-parasitic nematodes were found in 139 among 178 soil samples. Tylenchulus semipenetrans was collected from 85 out of 94 Citrus unshiu orchards examined (90.4%). C. junos orchards infested with T. semipenetrans were 60 out of 84 (71.4%). However, proportion of dominant species between the two was different. At domestic Citrus orchards, it proved that T. semipenetrans was the most important plant-parasitic nematode from this study. The overall frequency of the plant-parasitic nematodes was positively correlated with the cultivation years. More alkaline soils tended to harbor more T. semipenetrans. However the correlation coefficient between the two was very low.

This study was performed to investigate the escape of pine wood nematode (PWN), Bursaphelenchus xylophilus, from two vector species (Monochamus alternatus and M. saltuarius) through oviposition and feeding behavior. First, we checked number of PWNs escaped from M. alternatus emerged from three different cases of pine logs. In case A, healthy pine trees were cut into logs and left in pine forest infected with PWN. In case B, healthy pine trees were cut into logs, left in large screen cage, and let them oviposited by M. alternatus emerged from pine trees infested with PWN. In case C, pine trees which were harboring M. alternatus were cut into logs, and PWN was inoculated artificially. The M. alternatus adults emerged from the above three cases of pine logs were checked in the next year to know how many PWN they were harboring in their bodies. The percentages of M. alternatus harboring PWN (18.3 and 15.6%, respectively) and number of nematodes per vector (5,713.1±9,248.3 and 2,034.1±4,746.8 PWNs, respectively) in case A and B logs are similar to each other. However, the percentage and the number in case C (38.3% and 20,083.1±32,188.3 PWNs) were higher than those of case A and B. Among 52 M. alternatus adults harboring PWN from all the three cases, 20 adults (38.5%) were harboring more than 5,000 PWNs per beetle. And these 20 adults were harboring 97.9% of the total PWNs in 52 adults. Second, we checked the daily escape of PWNs from M. alternatus and M. saltuarius collected at pine forest infested with PWN. The PWN escaped from their vector body for 34.9±12.4 days for M. alternatus, and for 23.9±16.2 days for M. saltuarius, reaching at peak escape during the 2nd week of emergence of the two vector species. A 44.5 and 47.2% to the total PWNs escaped from vector body within 2 weeks of vector emergence for M. alternatus and M. saltuarius, respectively. The number of PWNs escaped from each vector was 3,570.6±5,189.2 and 1,556.2±1,710.3 for M. alternatus and M. saltuarius, respectively.

The toxicity of Kaempferia galanga rhizome materials and constituents against Meloidogyne incognita second‐stage juveniles (J2) and eggs were examined. The active principles of K. galanga rhizome were identified as the phenylpropanoids ethyl (E)‐cinnamate (EC, 1) and ethyl (E)‐p‐methoxycinnamate (EMC, 2) by spectroscopic analysis. Results were compared with those of carbofuran, fosthiazate, and metam‐sodium. In direct‐contact mortality bioassay, EC (LC50, 0.037 mg/ml) was the most toxic constituent, followed by EMC (0.041 mg/ml). EC was more effective than carbofuran (LC50, 0.092 mg/ml) but less active than fosthiazate (0.002 mg/ml). EC, egg hatch was inhibited 100, 93, and 87% at 125, 62.5, and 31.25 μg/ml, respectively. EMC caused 100, 81, and 75% inhibition of egg hatch at 125, 62.5, and 31.25 μg/ml, respectively. The inhibition of two phenylpropanoids were similar or more inhibition to that of either carbofuran or metam‐sodium but was lower than that of fosthiazate. In contact + fumigant mortality bioassay, EC and EMC treatments resulted in 86 and c 73% mortality at 0.5 and 0.125 mg/g soil, respectively. The lethality of these phenylpropanoids was almost similar to that of either carbofuran or metam‐sodium but was lower than that of fosthiazate. In vapor‐phase mortality bioassay, EC and EMC were more effective in closed container than open containers, indicating that the mode of delivery of these compounds was, in part, a result of vapor action. K. galanga rhizome‐derived materials, merit further study as potential nematicides and hatching inhibitors for the control of M. incognita populations.

There are increasing interests in developing methods specifically detecting pathogenic Bursaphelenchus xylophilus. In order to develop a detecting method for B .xylophilus, at first we generated monoclonal antibodies (MAbs) specific to B. xylophilus, discriminating from other pine tree resident nematodes. Among 2304 hybridoma fusions screened. We finally selected a MAb clone, 9F10 and used for further study. To identify the antigenic target of MAb-9F10, we employed several biochemical methods such as SDS-PAGE, 2 dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation to separate and isolate an antigenic target. Proteins from above methods were analyzed via nano-LC-ESI-Q-IT-MS. Peptides of GaLECtin were always detected from several proteomic analyses, suggesting that GaLECtin is the antigenic target of MAb-9F10.

Three acetylcholinesterase (ace) genes were identified from Bursaphelenchus xylophilus and named as Bxace1, Bxace2 and Bxace3. Open reading fragments of Bxace1, Bxace2 and Bxace3 were composed of 622, 625 and 588 amino acids, respectively. Sequencing comparison with Torpedo ace gene identified cholinebinding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that Bxace3 is more actively transcribed than Bxace1 (2-3 times) and Bxace2 (9-18 times) in both propagative and dispersal stages. The three ace genes were functionally expressed using baculovirus system. Kinetic analysis using three choline substrates demonstrated that BxAce2 has higher maximum velocity than BxAce1 (ca. 2 times) and BxAce3 (ca. 100 times) whereas BxAce3 shows lower Michaelis constant than BxAce1 (12.8 times) and BxAce2 (13.6 times). In inhibition assay using five organophosphates (OPs) and three carbamates (CBs), all the three BxAces were highly inhibited by paraoxon, DDVP and chlorpyrifos-oxon but not inhibited well by fenamiphos and fosthiazate. Interestingly, inhibition assay revealed that BxAce3 is less sensitive to all insecticides tested than other two BxAces. The inhibition kinetic data obtained in this study should provide essential information for the development of OP- and CB-based nematicidal agents againt B. xylophilus.

Nematicidal activity of aliphatic compounds was tested against pien wood nematode, Bursaphelenchus xylophilus. There was a significant difference in nematicidal activity among function groups. In a test with alkanols and 2-alkenols, compounds with C8-C11 chain length showed 100% nematicidal activity at 0.5 mg/mL concentration. C6-C10 2-alkenals exhibited >95% nematicidal activity, but the other compounds with C11-C14 chain length showed weak activity. Nematicidal activity of alkanoic acids with C7-C11 chain length was strong. Whole compounds belonging to hydrocarbons, alkanals and alkanoic acetate showed weak nematicidal activity at 0.5 mg/mL concentration. Nematicidal activity of compounds which showed strong nematicidal activity at 0.5 mg/mL concentration was tested at a lower concentration. At 0.25 mg/mL concentration, whole compounds except C8 alkanol, C8 2-alkenol and C7 alkanoic acid showed >80% nematicidal activity. C9-C11 alkanols, C10-C11 2-alkenols, C8-C9 2-alkenals and C9-C10 alkanoic acids showed >80% nematicidal activity at 0.125 mg/mL concentration. Only C11 alkanol exhibited strong nematicidal activity at 0.0625 mg/mL concentration