Morphometric Variation in Pine Wood Nematodes, Bursaphelenchus xylophilus and B. mucronatus, Isolated from Multiple Locations in South Korea

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1 Plnt Pthol. J. 29(3) : (2013) pissn eissn Note Open Aess The Plnt Pthology Journl The Koren Soiety of Plnt Pthology Morphometri Vrition in Pine Wood Nemtodes, Bursphelenhus xylophilus nd B. murontus, Isolted from Multiple Lotions in South Kore Yil-Sung Moon, Joung A Son* nd Chn Sik Jung Division of Forest Inset Pests nd Diseses, Kore Forest Reserh Institute, Seoul , Kore (Reeived on Septemer 14, 2012; Revised on Ferury 26, 2013; Aepted on Mrh 24, 2013) Intrspeifi vrition in morphometry of pine wood nemtodes Bursphelenhus xylophilus nd B. murontus in reltion to geogrphil lotions in South Kore ws investigted using morphometri hrters (ody length,, nd rtio, stylet length, nd spiule length for mle nemtode nd V (%) vlue for femle nemtode). B. xylophilus ws isolted from Pinus in Jinju (1998), Ulsn (2000), Yngsn (2000), Mokpo (2001) nd Jeju (2004), nd from P. in Gumi (2001). B. murontus ws isolted from P. in Jinju (1991) nd from P. in Milyng (2001). The ody length of mle nd femle B. xylophilus hd the highest oeffiient of vriility nd showed signifint differenes mong geogrphil lotions. The V (%) vlue for femle B. xylophilus showed the lowest oeffiient of vriility, hnging little with geogrphil re nd host plnt. All morphometri hrters in B. murontus exept for stylet length nd femle ody length showed no signifint differenes etween lotions or hosts, suggesting they my not e ffeted y geogrphil re or host plnt. Keywords : geogrphil lotion, intrspeifi vrition, pine wilt disese, Pinus, Pinus Outreks of pine wilt disese in Est Asin ountries re used y the exoti pine wood nemtode, Bursphelenhus xylophilus (Steiner et Buhrer) Nikle, whih ws previously endemi to North Ameri. In South Kore, pine wilt disese ws first disovered in pine forest in Busn ity in 1988, following introdution of this nemtode, nd hs sine spred ross the ountry. The spred of the nemtode hs een filitted y the vetor Monohmus lterntus Hope in the southern prt of South Kore (Yi et l., 1989), nd y M. slturius Geler in northern prt of South Kore (Kim et l., 2007). Following its initil disovery in South Kore, B. xylophilus hs sine een deteted in ded pine trees in the lotions of Jinju (1998), Ulsn (2000), Yngsn *Corresponding uthor. Phone) , FAX) E-mil) joung_son@hotmil.om (2000), Mokpo (2001), Gumi (2001), Jeju (2004), Seoul (2007), Yeosu (2010), nd y 2011 it hd spred to 44 other lotions (Choi et l., 2012). This pthogeni pine wood nemtode hs lso een introdued to the Europen Union vi numerous soures, inluding round wood, pkging, nd wood hips imported from infeted res (Evns et l., 1996). Brsh (2001) nlyzed five morphometri hrters of the genus Bursphelenhus olleted in the Europen Union, nd showed tht B. xylophilus nd B. murontus Mmiy nd End exhiited wide vrition in morphometri hrters suh s ody length. In Chin, Bursphelenhus spp. inluding B. xylophilus were deteted from imported wooden pkging in Ningo Port (Gu et l., 2008). Yin et l. (1988) nd Yusheng et l. (2002) reported tht some essentil morphometri hrters of the genus Bursphelenhus olleted in Hunn nd Gungdong provine in Chin, respetively. Morphometri hrters of the plnt pthogeni nemtode Prtylenhus penetrns Filip. nd Shuurm-Stekh, suh s ody length, ody width, esophgus length, stylet length, nd rtio, n vry ording to vriety of environmentl ftors, inluding host plnt (Trte nd Mi, 1976). Townshend (1991) reported tht the morphologil hrteristis of P. penetrns were ffeted y the host to greter extent thn y geogrphil re. Of those vrile morphometri hrters, the V (%) vlue of P. penetrns ws insuffiiently ffeted y host plnt for it to e of dignosti vlue (Trte nd Mi, 1976). Trjn nd Frederik (1978) reported tht the stylet lengths of P. rhyurus nd P. offee hd the lowest vriility of the hrters studied. In the se of Meloidogyne renri (Nel) Chitwood, the vlues of most morphometri hrters were signifintly different etween popultions (Cliff nd Hirshmnn, 1985). Here, we investigted the intrspeifi vrition of morphometri hrters of B. xylophilus nd B. murontus from different geogrphil lotions nd host plnts to estlish the extent of morphologil vrition in popultions of these two speies in South Kore.

2 Morphometri Vrition in Pine Wood Nemtodes in South Kore 345 Mterils nd Methods The pine wood nemtode, B. xylophilus, ws Pine wood nemtodes, B. xylophilus, were isolted from ded pine trees (Pinus Prl.), infeted with pine wilt disese, in Jinju, Ulsn, Yngsn, Mokpo, nd Jeju, nd from P. Sie. et Zu. in Gumi. B. murontus ws similrly isolted from P. in Jinju nd from P. in Milyng, South Kore (Fig. 1). Severl dult nemtodes were rndomly olleted from vrious stges of nemtodes whih were extrted from severl ded trees t eh lotion. All olleted pine wood nemtodes were fixed with F.G. 4:1 solution (80% formlin nd 20% glyerin), whih ws heted to 80 o C, nd then trnsferred to nhydrous glyerin to prepre permnent speimens using modified Seinhorst s rpid glyerin method (Seinhorst, 1959). Body length, mximl ody dimeter, distne from nterior end to phrynx-intestine juntion, til length, stylet length, spiule length nd distne from nterior end to vulv of eh pine wood nemtode were mesured. Morphometri hrter of rtio, rtio nd rtio were lulted from ody length divided y mximl ody dimeter, y distne from nterior end to phrynx-intestine juntion nd y til length, respetively. V (%) vlue ws lso lulted from perentge of distne from nterior end to vulv divided y ody length. For eh gender of B. xylophilus, the morphometri hrters of B. xylophilus were seprtely ompred mong the six lotionl groups using ANOVA nd Tukey s honestly signifint differene method. The morphometri hrters of B. murontus were ompred etween lotionl groups using 2-tiled t-test (P < 0.05). Results nd Disussion The morphometri fetures of mle nd femle B. xylophilus in eh lotionl re re shown in Tles 1 nd 2, respetively. The men ody length of mle B. xylophilus from the six lotions ws divided into three sugroups: Gumi, Jeju, nd the other four lotions. The men ody length of mle B. xylophilus from the Gumi lotion ws the longest mong the six lotionl groups, with the Jeju lotion nemtodes eing the seond longest. In the other four lotions (Jinju, Ulsn, Yngsn, nd Mokpo), the men ody length of mle B. xylophilus did not signifintly differ from eh other. The men ody length of femle B. xylophilus ws divided into the sme three sugroups s for mles. The men ody length of the six lotionl groups were smller thn those from the Busn lotion in oth mles nd femles (Choi nd Moon, 1989), ut overlpped with the men ody length of mle nd femle B. xylophilus in Jpn tht were 730 µm nd 810 µm, respetively Fig. 1. The lotions where pine wood nemtodes were olleted in South Kore. Bursphelenhus xylophilus ws olleted from Jinju, Ulsn, Yngsn, Mokpo, Gumi, nd Jeju. Est Asin types of B. murontus ws olleted from Jinju nd Milyng. (Mmiy nd Kiyohr, 1972). The men ody length lso overlpped with those of nemtodes found in Jpnese pkging wood in Chin, whih were 870 µm nd 1021 µm, respetively (Li et l., 2009). Our dt flls within the rnge of mle nd femle B. xylophilus ody lengths tht were µm nd µm, respetively, in the report of Brsh (2001). There ws wide vrition of B. xylophilus ody length. In se of P. penetrns, wide vrition of ody length n e explined y host plnts or the geogrphil origin of the nemtode popultions (Trte nd Mi, 1976; Townshend, 1991). A noteworthy feture of the Gumi lotionl group is tht oth mle nd femle B. xylophilus hd the longest ody length nd were isolted from P., wheres the other nemtodes were isolted from P.. Trte nd Mi (1976) showed tht nemtode ody length ws influened y host plnts, thus the ody length of B. xylophilus in Gumi my e relted to its host plnt. A unique spet of the Jeju lotionl group is tht Jeju is n islnd found long the Koren Peninsul (Fig. 1), nd this feture might e relted to the ft tht the ody lengths of oth mle nd femle B. xylophilus in the Jeju lotion were the seond longest mong the nemtodes we nlyzed. The oeffiient of vriility for ody length ws 1.55 nd this ws notiely the lrgest mong the six

3 346 Moon et l. Tle 1. Morphometri dt of mle Bursphelenhus xylophilus olleted in South Kore. Men ± SD re shown with the rnge of mesurement in prentheses Lotion Host plnt Colletion yer Spiule Numer of nemtode Jinju Pinus ± ± ± ± ± ±2.1 5 ( ) ( ) ( ) ( ) ( ) ( ) Ulsn Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) Yngsn Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) Mokpo Pinus ± ± ± ± ± ±3.7 5 ( ) ( ) ( ) ( ) ( ) ( ) Gumi Pinus ± ± ± ± ± ±4.3 7 ( ) ( ) ( ) ( ) ( ) ( ) Jeju Pinus ± ± ± ± ± ±1.2 7 ( ) ( ) ( ) ( ) ( ) ( ) Busn Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) CV Men numers followed y sme letters in eh olumn re not signifintly different t the 5% level y Tukey s honestly signifint differene method. Dt from Choi nd Moon (1989). CV vlues do not inlude the dt from Busn. Tle 2. Morphometri dt of femle Bursphelenhus xylophilus olleted in South Kore. Men ± SD re shown with the rnge of mesurement in prentheses Lotion Host plnt Colletion yer V (%) Numer of nemtode Jinju Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) Ulsn Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) Yngsn Pinus ± ± ± ± ± ±1.3 8 ( ) ( ) ( ) ( ) ( ) ( ) Mokpo Pinus ± ± ± ± ± ±1.0 7 ( ) ( ) ( ) ( ) ( ) ( ) Gumi Pinus ± ± ± ± ± ±2.2 8 ( ) ( ) ( ) ( ) ( ) ( ) Jeju Pinus ± ± ± ± ± ±1.5 9 ( ) ( ) ( ) ( ) ( ) ( ) Busn Pinus ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) CV Men numers followed y sme letters in eh olumn re not signifintly different t the 5% level y Tukey s honestly signifint differene method. Dt from Choi nd Moon (1989). CV vlues do not inlude the dt from Busn. morphometri hrters mesured. This suggests tht B. xylophilus ody length is the most vrile morphometri hrter in this study, despite the ft tht the four lotionl groups of Jinju, Ulsn, Yngsn nd Mokpo were not signifintly differentited from one nother. The geogrphil vrition of pine wood nemtode ody length, however, seems not to e relted to geneti vrition s the nemtode popultion in Ulsn groups in the sme sulde of Busn, rther thn tht in Jinju, whih is loted in seprte lde (Jung et l., 2010). The men rtios of mle nd femle B. xylophilus ross the six lotions were not signifintly different from

4 Morphometri Vrition in Pine Wood Nemtodes in South Kore 347 eh other. The rtio of B. xylophilus in this study ws mong the lest vrile morphometri hrters. In the study of Li et l. (2009), however, the rtio of three isoltes of femle B. xylophilus from Jpn, Cnd nd Chin ws useful hrter for delineting popultions. It n e supposed tht the geogrphi distnes etween six lotionl res in South Kore were not suffiiently sustntil to result in lotionl differentition of the rtio. The men rtio of mle B. xylophilus were not suffiiently vrile to identify lotionl groups in this study. The men rtio of femle B. xylophilus from the Gumi lotion ws signifintly smller thn those of other five lotionl groups, nd the host plnt of B. xylophilus from the Gumi lotion ws P., wheres those from other five lotionl groups were P.. Bsed on this result, the men rtio of femle B. xylophilus seems to hve reltion to speies of host plnts. The men rtio of mle B. xylophilus from the Jeju lotion ws signifintly lrger thn tht of other five lotionl groups. Bsed on this result, the men rtio of mle B. xylophilus seems to hve reltion to lotionl re. The men rtio of femle B. xylophilus, however, ws not suffiiently vrile to identify lotionl groups in this study, wheres Li et l. (2009) found it served s nother useful hrter for seprting popultions. length of B. xylophilus is known s useful hrter for popultion identifition (Li et l., 2009), while tht of some Prtylenhus spp. is known s the lest vrile hrter for speies identifition (Romn nd Hirshmnn, 1969; Trjn nd Frederik, 1978). In this study men stylet lengths of mle B. xylophilus did not seprte the lotionl groups. In ontrst, the men stylet lengths of femle seprted signifintly the lotionl groups into three sugroups: Gumi, Ulsn, nd the other four lotions. The men spiule length of mle B. xylophilus of the Jeju lotionl group ws signifintly shorter thn tht of other five lotionl groups. Aordingly, this morphometri hrter hs een used for popultion identifition (Li et l., 2009). V (%) vlues of femle B. xylophilus ross the six lotionl groups were not signifintly different from eh other, nd hve the lowest oeffiient of vriility mong the six morphometri hrters nlyzed in this study. These results suggest tht V (%) vlue is minimlly ffeted y geogrphil lotions nd host plnts. A similr Tle 3. Morphometri dt of mle Bursphelenhus murontus olleted in South Kore. Men ± SD re shown with the rnge of mesurement in prentheses Lotion Host plnt Colletion yer Spiule Jinju Pinus ±73.4 ( ) 49.6±7.7 ( ) 11.9±1.3 ( ) 21.1±2.5 ( ) 15.3±1.6 ( ) 21.4±1.5 ( ) Milyng Pinus ± ± ± ± ± ±2.0 ( ) ( ) ( ) ( ) ( ) ( ) Jeju Pinus ± ± ± ± ± ±7.0 ( ) ( ) ( ) ( ) ( ) ( ) CV Men numers followed y sme letters in eh olumn re not signifintly different t the 5% level y 2-tiled t-test. Dt from Choi nd Moon (1989). CV vlues do not inlude the dt from Jeju. Numer of nemtode Tle 4. Morphometri dt of femle Bursphelenhus murontus olleted in South Kore. Men ± SD re shown with the rnge of mesurement in prentheses Lotion Host plnt Colletion yer Jinju Pinus ±52.0 ( ) 49.5±6.0 ( ) 12.5±1.1 ( ) 20.6±2.0 ( ) 14.8±1.5 ( ) 73.2±2.3 ( ) Milyng Pinus ± ± ± ± ± ±2.4 ( ) ( ) ( ) ( ) ( ) ( ) Jeju Pinus ± ± ± ± ± ±1.2 ( ) ( ) ( ) ( ) ( ) ( ) CV Men numers followed y sme letters in eh olumn re not signifintly different t the 5% level y 2-tiled t-test. Dt from Choi nd Moon (1989). CV vlues do not inlude the dt from Jeju. V (%) Numer of nemtode

5 348 Moon et l. pttern ws oserved in six speies of Prtylenhus, where V (%) vlue hd the lowest oeffiient of vriility (Romn nd Hirshmnn, 1969). In ddition, Trte nd Mi (1976) found tht V (%) vlue of P. penetrns ws not suffiiently ffeted y host plnts to e of dignosti vlue. The morphometri fetures of ody length, nd rtio, stylet length, nd spiule length of mle B. xylophilus nd those of ody length, nd rtio, nd stylet length of femle B. xylophilus isolted from Jeju were signifintly different from those of Gumi. These differenes might e indued (or mintined) y the se rrier s Jeju is n isolted islnd, nd my lso e due to differenes in host plnts. In the se of B. murontus, mle stylet lengths nd femle ody lengths showed signifint differenes etween the Jinju nd Milyng lotionl groups (Tles 3 nd 4). The men ody length of the Jinju nd Milyng lotionl groups were smller thn those from Jeju in oth mles nd femles (Choi nd Moon, 1989), ut overlpped with those of B. murontus in Jpn (Mmiy nd End, 1979). The rtios of, nd in mles nd femles, the spiule length of mles, nd the V (%) vlue for femles of B. murontus did not differ etween the two lotionl groups, suggesting tht these hrters re minimlly ffeted y geogrphi lotions or host plnts. Bsed on the study of Li et l. (2009), the three popultions of oth mle nd femle B. murontus (Est-Asin type) were lso not seprted y five different morphometri hrters. The V (%) vlue hd the lowest oeffiient of vriility mong the six morphometri hrters, suggesting it might e useful for speies identifition. However, there ws no signifint differene (P < 0.05, 2-tiled t-test) etween the V (%) vlue of B. murontus nd tht of B. xylophilus in this study nd it is well known tht these two speies re morphologilly too similr to distinguish eh other exept the shpe of til terminus (Brsh, 2008; Mmiy nd End, 1979). The morphometri hrters of mle B. xylophilus suh s the rtio nd spiule length were ffeted y lotionl re rther thn host plnt, while tht of femle B. xylophilus suh s the rtio ws ffeted y host plnt rther thn lotionl re. The ody length of B. xylophilus ws ffeted y lotionl re, host plnt, or oth. In this study, it ws onfirmed tht some morphometri hrters of B. xylophilus were ffeted y lotionl re, host plnt, or oth nd those hrters showed extended rnge of vrition. When using these hrters in txonomi studies, we should e reful of the mount of vrition of those hrters. Referenes Brsh, H Bursphelenhus speies in onifers in Europe: distriution nd morphologil reltionships. EPPO Bulletin 31: Brsh, H The enlrgement of the xylophilus group in the genus Bursphelenhus. In: Pine wilt disese: worldwide thret to forest eosystems. ed. y Mot, M. M. nd Vieir, P, pp Springer Siene, Business Medi BV, Berlin, Germny. Choi, W. I., Go, S. H. nd Lee, S. K Report of monitoring for forest insets nd disese in Kore. Kore Forest Reserh Institute, Seoul, Kore. (in Koren) 189pp. Choi, Y. E. nd Moon, Y. S Survey on distriution of pine wood nemtode (Bursphelenhus xylophilus) nd its pthogeniity to pine trees in Kore. Koren J. Plnt Pthol. (in Koren) 5: Cliff, G. M. nd Hirshmnn, H Evlution of morphologil vriility in Meloidogyne renri. J. Nemtol. 17: Evns, H. F., MNmr, D. G., Brsh, H., Chdoeuf, J. nd Mgnusson, C Pest Risk Anlysis (PRA) for the territories of the Europen Union (s PRA re) on Bursphelenhus xylophilus nd its vetors in the genus Monohmus. EPPO Bulletin 26: Gu, J., Zhng, J., Chen, X., Brsh, H. nd Burgermeister, W Bursphelenhus spp. in Wood Pkging Interepted in Chin. In: Pine wilt disese: worldwide thret to forest eosystems. ed. y Mot, M. M. nd Vieir, P., pp Springer Siene, Business Medi BV, Berlin, Germny. Jung, J., Hn, H., Ryu, S. H. nd Kim, W Mirostellite vrition in the pinewood nemtode, Bursphelenhus xylophilus (Steiner nd Buhrer) Nikle in South Kore. Genes Genomis 32: Kim, K. H., Seo, S. T., Oh, E. S., Lee, S. H., Lee, S. K. nd Shin, S. C Diseses of onifers in Kore. Kore Forest Reserh Institute, Seoul, Kore. (in Koren) 137pp. Li, H., Trinh, P. Q., Weyenerge, L. nd Moens, M Chrteristion of Bursphelenhus spp. isolted from pkging wood imported t Nnjing, Chin. Nemtology 11: Mmiy, Y. nd End, N Bursphelenhus murontus n. sp. (Nemtod: Aphelenhoidide) from pine wood nd its iology nd pthogeniity to pine trees. Nemtologi 25: Mmiy, Y. nd Kiyohr, T Desription of Bursphelenhus ligniolus n. sp. (Nemtod: Aphelenhoidide) from pine wood nd histopthology of nemtode-infested trees. Nemtologi 18: Romn, J. nd Hirshmnn, H Morphology nd morphometris of six speies of Prtylenhus. J. Nemtol. 1: Seinhorst, J. W A rpid method for the trnsfer of nemtodes from fixtive to nhydrous glyerin. Nemtologi 4: Trjn, A. C. nd Frederik, J. J Intrspeifi morphologil vrition mong popultions of Prtylenhus rhyurus nd P. offee. J. Nemtol. 10: Trte, R. nd Mi, W. F Morphologil vrition in Prtylenhus penetrns. J. Nemtol. 8: Townshend, J. L Morphologil oservtions of Prtylen-

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