Short Communication

Nematology, 2011, Vol. 13(3), 377-380 Short communication

Nematicidal activity of Korean domestic plant extracts against thepinewoodnematode,Bursaphelenchus xylophilus ∗ Eunae KIM, Junheon KIM and Il-Kwon PARK

Pine wilt disease, caused by the pine wood nematode, (31), twig (three) and fruit (two), were collected during Bursaphelenchus xylophilus, has been considered as an in- June-September in 1998-2000. The economic importance ternational problem in Japan, China, Taiwan and Korea of these plants is described in detail elsewhere (Lee, 1982; in Far East Asia, Mexico in Southern America and Por- Kim, 1996). They were dried in an oven at 60◦Cfor tugal in Europe (Shin & Han, 2006). This disease was 2 days and finely powdered using a blender. Each sample first reported in Korea in Busan City (Yi et al., 1989); (100 g) was extracted twice with 500 ml methanol at since then, it has spread to several areas of the penin- room temperature and filtered (Toyo filter paper No. 2, sula (Chung, 2002). Control of this disease depends pri- Toyo Roshi, Tokyo, Japan). The combined filtrate was marily on commercial nematicides or insecticides such as concentrated in vacuo at 35◦C using a rotary vacuum abamectin and emamecin, benzoate and thiacloprid (Shin evaporator (EYELA, JP/N-1000VW, Tokyo Rikakikai, & Han, 2006). Although the application of synthetic pes- Tokyo, Japan). For screening the nematicidal activity of ticides is effective, adverse effects on the environmen- plant extracts, the extract was suspended in distilled water tal and human health are of concern. Thus, there is a containing Triton X-100 (5 mg ml−1). Test solutions need to search for sustainable alternatives. Phytochemi- (2 μl) were filled into each well of a 96-well plate (Falcon) cals may provide alternatives because many plant extracts containing 50-150 nematodes in 98 μl water/Triton X- and their constituents are known to possess nematicidal 100 solution. Thus total volume of the solution in a activity against a wide range of plant-parasitic nematodes well was 100 μl. The primary concentration of tested (Chitwood, 2002; Choi et al., 2006). Although nematici- plant extracts in a well was 10 mg ml−1. Nematicidal dal activities of many plant essential oils and their con- activity of plant extracts with >80% mortality were stituents against B. xylophilus have been reported (Park et tested at lower concentration. Control wells contained the al., 2005, 2007; Choi et al., 2007a, b, c; Kim et al., 2008; water/Triton X-100 solution in the same volume as the Seo et al., 2010), little work has been done on solvent ex- test solutions. Three trials on different days were used to tracts of Korean domestic plant species (Choi et al., 2006). test the nematicidal activity with four replicates per trial. In this study, we investigated the nematicidal activities of The tested plates were stored under 14/10 h (light/dark) 102 Korean domestic plant extracts, which have not been light regime at 25 ± 1◦C and 60% relative humidity. tested previously against B. xylophilus. Nematode mortality was recorded after 48 h treatment Bursaphelenchus xylophilus was isolated from chips of as follows: 10 μl of the test suspension was transferred infected pine wood collected in Haman-region (on March to 100 μl fresh water with micropipette and observed 2004), Gyoungsangnam-do province, Korea and extracted under a microscope. One minute after transfer, it was by the Baermann funnel method (Chawal & Parasad, observed whether the nematodes were dead. Nematodes 1975; Park et al., 2005). The colony was maintained on a were classified as dead if their bodies were motionless lawn of Botrytis cinerea cultured on potato dextrose agar and straight. Nematode mortality was adjusted for control ◦ medium (PDA) in the dark at 28 C. A total 102 samples mortality using Abbott’s correction (1925); Mc = ((Mo − of 58 plant species from 30 families were tested in this Me)/(100 − Me)) × 100, where Mo is mortality of study. Different tissues, namely bark (25), leaf (41), wood treated nematodes (%), Me mortality of control (%) and

Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, South Korea ∗ Corresponding author, e-mail: [email protected] Received: 13 September 2010; revised: 1 November 2010 Accepted for publication: 1 November 2010 Keywords: biological control, natural extracts, pine wilt.

Mc corrected mortality (%). Nematode mortality was raceae, Myricaceae, Pinaceae, Rosaceae, Taxodiaceae, transformed to arcsine square root values for analysis of Theaceae and Ulmaceae showed strong nematicidal activ- variance (ANOVA). Treatment means were compared and ity against pine wood nematode. Park et al. (2005, 2007) separated by Tukey-Kramer HSD test (P = 0.05). All already reported that essential oils from Cinnamomum calculations were performed with JMP program (SAS, verum and Litsea cubeba of Lauraceae revealed strong Cary, NC, USA). nematicidal activity against pine wood nematode. Solvent Nematicidal activity of plant extracts with >50% mor- extract of Forsythia koreana revealed 81.4% nematicidal tality was recorded (Table 1). Nematicidal activity of activity against pine wood nematode in this study, but es- plant extracts varied according to plant species and plant sential oils of this plant show weak nematicidal activity in part. Of the 102 plant extracts, 42 plant extracts in a previous study (Choi et al., 2007b). This might be at- 22 families showed >50% nematicidal activity against tributed to the test concentration and difference of chem- pine wood nematode at 10 mg ml−1 concentration. The ical composition of solvent extracts and essential oil. Ne- other 60 plant extracts (13 bark extracts, 30 leaf ex- maticidal activity of wood or bark extract was generally tracts, 14 wood extracts, two twig extracts and one fruit stronger than leaf extract in this study. This result indi- extract) showed weak nematicidal activity. Among 31 cated that wood or bark extracts could be good candidates wood extracts, strong nematicidal activity (100%) was for finding nematicidal compounds from higher plants obtained with extracts of Neolitsea aciculata, Prunus against pine wood nematode. Based upon our data and sargentii and Rhus chinensis at 10 mg ml−1 concen- these earlier findings, some plants described might be use- tration. Extracts from Camellia japonica, Carpinus cor- ful products for developing new types of nematicides or data, Daphniphyllum macropodum, Eucommia ulmoides, biorational management agents for controlling pine wood Ilex cornuta, Myrica rubra, Vaccinium bracteatum and nematode, although their effects on natural enemies or the Zelkova serrata showed strong nematicidal activities (80- environment have not been fully investigated. 99%). The other samples exhibited activities <80%. In a test with leaf extracts, strong nematicidal activity was observed in extract of Machius japonica (100%) fol- References lowed by Picea koraiensis (99.4%), Meliosma oldhamii (82.7%), and Zelkova serrata (81.0%). The other 37 plant ABBOTT, W.S. (1925). A method of computing the effective- species gave <80% mortality. Strong nematicidal activ- ness of an insecticide. Journal of Economic Entomology 18, ity (>80%) was observed from bark extract of Carpinus 265-267. cordata, Comus kousa, Cryptomeria japonica, Machius CHAWAL,M.L.&PARASAD, S.K. (1975). Techniques in japonica, Quercus glauca and Zelkova serrata. Nemati- nematology. II. Comparative efficiency of sampling tools and cidal activity of the other 19 bark extracts was less than nematode extraction methods. Indian Journal of Nematology 4, 115-123. 80%. Twig extract of Smilax china and fruit extract of CHITWOOD, D.J. (2002). Phytochemical-based strategies for Forsythia koreana showed 86.6 and 81.4% nematicidal . Annual Review of Phytopathology −1 nematode control 40, 221- activity at 10 mg ml concentration, respectively. Plant 249. extracts which showed >80% nematicidal activity were CHOI, I.H., PARK, J.Y., SHIN,S.C.&PARK, I.K. (2006). −1 tested at lower concentrations. At 5 mg ml concentra- Nematicidal activity of medicinal plant extracts and two cin- tion, extracts from Rhus chinensis (wood), Prunus sar- namates isolated from Kaempferia galanga L. (Proh Hom) gentii (wood), Meliosma oldhamii (leaf) and Zelkova ser- against the pine wood nematode, Bursaphelenchus xylophi- rata (wood) exhibited >80% nematicidal acitivty. The lus. Nematology 8, 359-365. other samples showed moderate or weak nematicidal ac- CHOI, I.H., KIM,J.,SHIN,S.C.&PARK, I.K. (2007a). Ne- tivity. At 2.5 mg ml−1 concentration, Meliosma oldhamii maticidal activity of monoterpenoids against the pine wood showed the strongest nematicidal activity followed by nematode (Bursaphelenchus xylophilus). Russian Journal of Nematology 15, 35-40. Rhus chinensis, Prunus sargentii and Zelkova serrata. CHOI, I.H., PARK, J.Y., SHIN, S.C., KIM,J.&PARK,I.K. In the laboratory study with 102 samples against pine (2007b). Nematicidal activity of medicinal plant essential oils wood nematode, the responses varied with plant species against the pinewood nematode (Bursaphelenchus xylophi- and plant part sampled. Among plant species tested, lus). Applied Entomology and Zoology 42, 397-401. plant species belonging to Anacardiaceae, Betulaceae, CHOI, I.H., SHIN,S.C.&PARK, I.K. (2007c). Nematicidal Erieaceae, Eucomiaceae, Euphorbiaceae, Fagaceae, Lau- activity of onion (Allium cepa) oil and its components against

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