• Journal of Korean Society of Forest Science
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• v.94 no.5 s.162
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• pp.334-337
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• 2005

Pine wood nematode isolates from Haman, Gyoungsangnam-do were cryopreserved with three different cryoprotectants (glycerol, ethylene glycol and dimethylsulfoxide), Nematode Growth medium (NGM) and M 9 buffer solution, respectively, and then survival rate, reproduction ability, and pathogenicity of stored nematodes were compared. Survival rates of juvenile was excellent in 15% glycerol solution as $72{\pm}5.3%$. The survival ability of nematodes at juvenile stage was more efficient than that of nematodes at adult stage. Pre-incubation treatment at $4^{\circ}C$ before storing in liquid nitrogen didn't affect the survival ability of pine wood nematode. When the reproduction ability of nematodes was confirmed on Botrytis cinerea agar plates at $28^{\circ}C$ incubator, there was no difference between cryopreserved and non-cryopreserved. Pathogenic ability of cryopreserved nematodes was also evaluated and confirmed by artificially inoculating them on 2-year old red pine seedlings.

• Research in Plant Disease
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• v.18 no.2
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• pp.101-108
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• 2012

In 2006, pinewood nematode, Bursaphelenchus xylophilus, was isolated from about 50 years old trees of Pinus densiflora and P. koraiensis showing leaf-wilt and -drying symptoms in Gwangju, Gyeonggi-do and Chuncheon, Gangwon-do. Isolation of pinewood nematodes from sapwood of infected pine trees showed no difference in population density between tree species and among the sampling heights on the main stem. Migration of pinewood nematodes in the host tree were investigated by inoculation of red pine (P. densiflora, 3 years old) seedlings with B. xylophilus. The nematodes seemed to move in red pine seedlings prior to multiplication and it might have taken about 20 days to start multiplication and expression of symptoms including wilt and dieback. In initial time after inoculation, nematodes started migration through the cortical resin canal from inoculated site and further showed upward and downward movements. More nematodes were observed in cortical resin canal during early period of inoculation and later in resin canal of xylem and tracheid also while, the pith still remained free from nematode. The density of B. xylophilus was higher in seedlings of low-vigor with poor root growth than in seedlings of normal root growth. Seedlings showing high density of B. xylophilus exhibited stem discoloration and secondary infection by fungus at the inoculation site.

• Journal of Korean Society of Forest Science
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• v.97 no.6
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• pp.561-564
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• 2008

Pinus densiflora(red pine) stands in Korea have been faced with the serious threat by pine wilt disease caused by Bursaphelenchus xylophilus (nematodes). It is not easy to early detect and prevent the infected trees because those cannot be visually identified during the initial phase of infection. Red pine is usually infected by B. xylophilus from May to July and can be just visually detected in October or November. While the infected trees are wilted, the spectral value of Near Infrared (NIR) is supposed to be decreased. Based on this phenomena, in this paper, the vegetation vitality change of infected trees was analyzed using vegetation indices. Spectral values of Red, Green and NIR had been acquired monthly by a portable NIR camera in the same place of red pine stands infected by pine wilt disease. It could be proven that the vegetation index, or vegetation vitality of damaged trees starts to decrease from June, in the early infecting phase.

• Molecules and Cells
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• v.26 no.1
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• pp.81-86
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• 2008

RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products.

• Journal of Microbiology and Biotechnology
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• v.29 no.7
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• pp.1117-1123
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• 2019

Control of pine wilt disease, which is caused by pine wilt nematode Bursaphelenchus xylophilus, is heavily dependent on the use of chemicals such as abamectin. Although such chemicals are highly effective, demands for alternatives that are derived preferentially from natural sources, are increasing out of environmental concerns. One of the challenges to discovery of alternative control agents is lack of fast and efficient screening method that can be used in a high-throughput manner. Here we described the development of colorimetric assay for the rapid and accurate screening of candidate nematicidal compounds/biologics targeting B. xylophilus. Contrary to the conventional method, which relies on laborious visual inspection and counting of nematode population under microscope, our method utilizes a redox dye that changes its color in response to metabolic activity of nematode population in a given sample. In this work, we optimized parameters of our colorimetric assay including number of nematodes and amount of redox dye, and tested applicability of our assay for screening of chemicals and biologics. We demonstrated that our colorimetric assay can be applied to rapid and accurate quantification of nematode viability/mortality in a nematode population treated with candidate chemicals/biologics. Application of our method would facilitate high-throughput endeavors aiming at finding environment-friendly control agents for deadly disease of pine trees.

• The Plant Pathology Journal
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• v.24 no.2
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• pp.178-182
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• 2008

Five essential oils and 15 herbal extracts were evaluated to control Bursaphelenchus xylophillus in laboratory. The essential oils from clove plant (Syzygium aromaticum), mustard (Brassica integrefolia), thyme (Thymus vulgaris), and Pelargonium inquinans were found to be highly promising and gave excellent control of the nematodes at all the time of exposure. Among them, the least one gave 91.3% mean mortality rate at 24 hours of exposure time, which is highly significant from the control. While in the second study, most of the methanol (Desmodium caudatum, Paulownia coreana, Auckulandia lappa, Sophota flavescens, Aloe sp., Rheum palmatum, Zingiber officinale, Magnolia officinalis, and Eugenia caryophyllata), hexane (Torreya nucifera, Pharbitis nil, Prunus mume, Melia azedarach, and Xanthium strumarium), and hot water (Cinnamomum cassia) herbal extracts killed the nematodes, but in varying degrees compared to the control. Only one extract was found to be promising viz Magnolia officinalis which found to be statistically different from the control and gave mean mortality of 72, 82.3, and 85.3 % for 24, 48, and 72 hours exposure, respectively. Further screening was conducted for M. officinalis with concentrations of 1,000, 100, and 10 ppm against the same species of nematode with the same time of exposure. However, it gave an excellent result for 1,000 ppm for all time of exposure, whereas for the 100 and 10 ppm it gave mean mortality of 39.5 and 25.8% for the time 72hrs, respectively that were statistically different from the control.

• Korean journal of applied entomology
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• v.42 no.4
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• pp.307-313
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• 2003

Japanese pine sawyer, Monochamus alternatus, is a primary vector of pinewood nematode, Bursaphelenchus xylophilus in Korea. Emergence characteristics of the adults were studied in Jinju, Korea using dead pine logs in which the larvae of the sawyer overwintered from 1999 to 2002. Emergence data showed that the adults began to emerge out of the logs in sunny place from May the 15th in average of the four years. Cumulative emergence ratio (CER) reached at 50% at mid-June, and the emergence terminated by early or late July depending on years. In shady place, however,50% CER was reached 17 days later than in sunny place. The 50％ CER of males reached 1 to 4 days earlier than that of females depending on years. Of the adults emerged from the logs collected from February to April in 2001, 97.6％ was univoltine, and the rest was biennial which emerged from May to July of the next year. Female and male adults emerged throughout 24 hours; 32.3% of the total adults emerged from 8 to 12 a.m. It took 68.0 seconds for the adults to escape from pine logs.

• Journal of Korean Society of Forest Science
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• v.99 no.3
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• pp.337-343
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• 2010

The purpose of this study was to understand water stress development in pine seedlings inoculated with Bursaphelenchus xylophilus. Leaf water potentials of four years old Pinus densiflora seedlings inoculated with the nematode were measured with a plant moisture system at predawn and midday. The midday leaf water potentials on the 35th day after the inoculation were -1.04 MPa in the infected seedlings, while -0.94 MPa in the non-infected seedlings. The predawn water potentials on the 56th day were -0.71 MPa in the infected, while -0.26 MPa in the non-infected. At this time the leaves of the infected seedlings became yellow and resin did not exude on the cut basal stem. Density of the nematode was maximum on the 28th day, but dropped sharply on the 68th day. The phloem and pith tissues became brown and the resin exudation reduced in the infected seedlings. In conclusion, pinewood nematode inoculation developed water stress in pine seedlings in a month.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2004.05a
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• pp.135-135
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• 2004

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2002.11a
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• pp.147-147
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• 2002