Radopholus duriophilus sp. n. (Nematoda: Pratylenchidae) from Western Highland of Vietnam

Nematology 2003 Vol 5(4) 549-558

Radopholus duriophilus sp n (Nematoda Pratylenchidae)from Western Highland of Vietnam

Chau N NGUYEN 1 Sergei A SUBBOTIN 2 Mehrdad MADANI 3

Phap Q TRINH 1 and Maurice MOENS 34curren

1 Institute of Ecology and Biological Resources NCST 18 Hoang Quoc Viet Rd Hanoi Vietnam2 Institute of Parasitology RAS Leninskii Prospect 33 Moscow 177071 Russia

3 Agricultural Research Centre Burg Van Gansberghelaan 96 9820 Merelbeke Belgium4 Laboratory for Agrozoology Ghent University Coupure 555 9000 Ghent Belgium

Received 6 January 2003 revised 11 March 2003Accepted for publication11 March 2003

Summary ndash A new species of the genus Radopholus associated with durian (Durio zibetinus M) in the Western Highland of Vietnamis described as Radopholus duriophilus sp n The new species is close to R similis but is distinguished from R similis by the positionof the excretory pore located posterior to pharyngo-intestine junction (vs at level of pharyngo-intestine junction) oval shape sperm(vs rod-like) four incisures terminating far behind position of phasmid (vs three incisures terminating at or just behind phasmid) andbursa in male never reaching tail terminus (vs bursa reaching tail terminus) Females of R duriophilus sp n differ from R nativusfemales by stylet length (165-19 vs 19-23 sup1m) oval or kidney-shaped sperm (vs rod-like) four incisures at level of phasmid (vsthree) and their areolated lateral eld (vs not areolated) The position of excretory pore of both female and male is located posteriorto pharyngo-intestine junction (vs at level or anterior to pharyngo-intestine junction) Females of R duriophilus sp n differ from Rclarus females by stylet length (165-19 vs 19-21 sup1m) and areolated lateral eld (vs no areolation) Females of R duriophilus sp ndiffer from R musicola females by their lateral eld with equidistant incisures at mid-body (vs two deep outer folds and two faintshallow inner incisures) oval or kidney-shapedsperm (vs rod-like) and rounded terminus tail (vs sharply pointed) The species also spn differ in male stylet length (115-15 vs 88-12 sup1m) Females of R duriophilus sp n differ from R bridgei females by stylet length(165-19 vs 15-175 sup1m) median bulb length (11-165 vs 11-13 sup1m) length of hyaline tail (3-11 vs not more than 4 sup1m) and lateral eld areolated for entire body (vs not areolated except irregularly on neck and tail) The male differs by stylet length (115-15 vs 10-12sup1m) and length of the hyaline portion (4-9 vs 1-4 sup1m) In addition the relatively high level of ITS sequence divergence of the newspecies from R similis populations and the presence of nucleotide autapomorphies support a separate speci c status for these durianpopulations Results of surveys revealed that R duriophilus sp n is rather widely distributed in durian orchards and associated withdecline and death of trees in many durian nursery gardens Densities of nematode population reached thousands of individuals per g ofroot samples

Keywords ndash durian ITS molecular characterisationphylogeny rDNA RFLP taxonomy Vietnam

Ryss and Wouts (1997) classi ed 24 species withinthe genus Radopholus Thorne 1949 However Siddiqi(2000) accepted only 20 species placing several othersin other genera Since then only R musicola StantonMundo-Ocampo Baldwin amp Kaplan 2001 has been de-scribed in the genus Radopholus similis (Cobb 1893)Thorne 1949 is considered to be the most economicallyimportant species world-wide Recently R nativus Sher1968 R bridgei Siddiqi amp Hahn 1995 and R musicolaStanton Mundo-OcampoBaldwin amp Kaplan 2001 were

Corresponding author e-mail mmoensclofgovbe

reported as potential pests of wheat in West Australia (Ri-ley amp Kelly 2001) turmeric roots in Indonesia (Siddiqiamp Hahn 1995) and banana in North Australia (Stanton etal 2001) respectively Although R similis is prevalentin many tropical and subtropical regions throughout theworld it has never been recorded in Vietnam Earlier at-tempts to recover R similis from banana growing areasof Vietnam were unsuccessful (Eroshenko et al 1985Nguyen et al 1997 Van den Bergh et al 2000) How-ever in some neighbouring countries (Thailand Indone-

copy Koninklijke Brill NV Leiden 2003 549Also available online - wwwbrillnl

CN Nguyen et al

sia Malaysia and the Philippines) the species is commonand causes damage to banana and black pepper (Holde-man 1986 Razak 1994)

In 1998-1999many durian (Durio zibetinus M) grow-ers in Dak Lak provinceWestern HighlandVietnam wereconfrontedwith tree decline and death of young trees Fre-quently the problem was associated with imported plant-ing material Tree decline also appeared in some orchardsreplanted with the same breeding source In 1999 thePlant Quarantine Of ce of the Ministry of Agricultureand Rural Development (MARD) detected high popula-tion densities of a nematode in the rhizosphere and rootsof diseased durian but none or very few in unaffectedplants These nematodes met the description of the genusRadopholus (Siddiqi 2000) During 2000 and 2001 sur-veys were carried out in durian at numerous localities inDak Lak province The morphology morphometrics andmolecular data of the Vietnamese Radopholuspopulationsrevealed that these populations belong to a new specieswhich in this paper is described as Radopholus durio-philus sp n

Materials and methods

NEMATODE POPULATIONS

Sixteen farms were surveyed A total of 96 samplescomprising soil and roots were collectedNematodeswereextracted from soil by decantation followed by centrifu-gal otation and from roots by maceration and centrifu-gation (Coolen amp DrsquoHerde 1972) Three populations ofRadopholus (Buon Ma Thuot Curgma and Krong Ana)were successfully maintained on carrot discs (OrsquoBannonamp Taylor 1968) and used for further observations Fromtwo populations (Buon Ma Thuot and Krong Ana) about15 to 20 nematodes (juvenilesand adults) were transferredto 1 M NaCl for molecular observations The remainingnematodes were heat killed and xed in TAF (Seinhorst1959)

MORPHOLOGICAL STUDY

Fixed nematodes were processed and mounted in an-hydrous glycerine using the slow method of Hooper andEvans (1993) From each population morphometrics of30 females and 30 males were taken using a camera lucidadrawing tube attached to an Olympus CH40 light micro-scope

For scanning electron microscopy (SEM) specimenspreserved in anhydrous glycerine were transferred to a

drop of 4 formalin A subsequent ultrasonic treatment(10 min) removed particles adhering on the body surfaceof the specimen The nematodes were dehydrated bypassing them through a gradual ethanol gradient of 25(overnight) 50 75 95 (3 h each) and 100 (overnight)at 25plusmnC They were critical point dried with liquid CO2mountedon stubs and coated with gold-palladium(25 nm)before observation with a Jeol LSM-840 at 15 kV

MOLECULAR OBSERVATIONS

DNA extraction and ampli cation were made as de-scribed by Subbotin et al (2000) Primers rDNA1(50-TTGATTACGTCCCTGCCCTTT-30) and rDNA2 (50-TTTCACTCGCCGTTACTAAGG-3 0) (Vrain et al 1992)were used for ampli cation of the ITS regions includingthe 58S gene plus ankingareas of the 18S and 28S genesof rDNA Ampli ed product was puri ed using a QiagenGel Puri cation Kit (Qiagen GmbH Hilden Germany)PCR product was digested with one of eight restrictionenzymes viz AluI RsaI CfoI Bsp143I ScrFI Bsh1236ITru9I and TaqI (Promega Madison WI USA and MBIFermentas St Leon-Rol Germany) DNA fragments weresequencedusing primers rDNA1 rDNA2 and 58SM5 (50-GGCGCAATGTGCATTCGA-30) with a BigDye Termi-nator Cycle Sequencing Ready Reaction Kit (PE AppliedBiosystems Foster city CA USA) The resulting prod-ucts were puri ed using a Centri ex Gel Filtration Car-tridge (Edge Biosystems Inc Gaithersbugs MD USA)The DNA samples were sequenced by ABI Prism 377DNA Sequencer The DNA sequences of durian nematodepopulationswere aligned using ClustalX 164 (default op-tions) with six ITS-rDNA sequences of R similis fromGenBank The original ITS sequence of R duriophilussp n is deposited at GenBank under accession numbersAY257199 AY257200

Equally weighted maximum parsimony (MP) analysiswas performed using PAUP 40 beta version (Swofford1998) A heuristic search procedure was used with thefollowing settings ten replicates of random taxon addi-tion tree-bisection-reconnection branch swapping mul-tiple trees retained no steepest descent and acceleratedtransformation Gaps were treated as missing data Boot-strap analysis was calculated with 1000 replicates for MPtree Pair wise divergences between taxa were computedby PAUP as the absolute distance values and the percentmean distance values adjusted for missing data

550 Nematology

Radopholus duriophilus sp n from Vietnam

Fig 1 Radopholus duriophilus sp n Female A-F J A Anterior end including pharynx B Tail C Stylet D Vulva region andposterior branch of reproductive tract E Variation in tail tip morphology J Entire female body Male G-I K G Anterior endincluding pharynx H Tail I Variation in tail tip morphology K Entire male body F Sperm

Vol 5(4) 2003 551

CN Nguyen et al

Fig 2 SEM photographs of Radopholus duriophilus sp n Female A-E A Head lateral view B Head en face view C Vulva ventralview D Vulva lateral view and lateral eld E Tail phasmid and lateral incisures (arrow) Male F-J F Head lateral view G Headen face view H-I ventral view of spicules (arrow indicates direction of head) J Lateral view of tail (arrow indicates position ofphasmid)

552 Nematology

Radopholus duriophilus sp n from Vietnam

Fig 2 (Continued)

Radopholus duriophilus sp n

(Figs 1 2)

MEASUREMENTS

See Table 1

DESCRIPTION

Female

Body almost straight or slightly curved ventrally afterkilling by heat Cephalic region slightly set off and moreor less hemispherical with four or ve annules Labialdisc not distinct in LM hexagonal in SEM Laterallips terminating within second and third head annuleStylet moderately strong with rounded knobs dorsal

Speci c name meaning lsquowho likes durianrsquo

knob sometimes projected Cone slightly longer thanshaft plus knobs Rounded or oval median bulb welldeveloped Pharyngeal glands in tandem and forming along dorsally overlapping lobe Excretory pore locatedposteriorly to level of pharyngo-intestinal junction at adistance of half to full body diameter and zero to twoannules posterior to hemizonid hemizonid one to twobody annules wide Lateral eld completely areolated forentire body with four equidistant incisures Four incisuresat level of phasmid Three annules terminating at vulvaTwo genital branches equally developed spermathecaeround to oval and of equal size lled with small oval orkidney-shapedsperm Oocytes located in one or two rowsgenital branches sometimes reaching pharynxone or bothbranches may be re exed Postrectal intestine sac absentTail conical tapering and with shallow to deeply forkedtip terminus annulated rarely smooth narrow conoid

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CN Nguyen et al

Table 1 Morphometric characters of Radopholus duriophilus sp n Measurements in sup1m and in form mean sect standard deviation(range)

Buon Ma Thuot Curgma Krong Ana

Holotype Paratypes

Female Female Male Female Male Female Male

n 30 30 30 30 30 30L 622 603 sect 67 614 sect 34 588 sect 50 652 sect 54 669 sect 68 647 sect 30

(500-720) (550-667) (490-765) (555-750) (552-840) (585-690)Maximum body diam 28 23 sect 2 18 sect 2 19 sect 2 18 sect 2 23 sect 4 18 sect 2

(19-26) (16-22) (16-23) (14-22) (16-31) (14-22)Height of lip region 45 4 sect 02 6 sect 05 4 sect 04 6 sect 05 4 sect 07 6 sect 06

(35-42) (5-7) (3-5) (5-7) (3-6) (5-7)Diam of lip region 10 10 sect 06 85 sect 07 94 sect 06 8 sect 06 103 sect 08 82 sect 07

(9-11) (7-10) (8-11) (7-9) (8-115) (65-10)Length of stylet 18 177 sect 08 135 sect 09 174 sect 08 13 sect 07 18 sect 07 136 sect 11

(165-19) (12-15) (165-19) (12-14) (17-19) (115-15)Width of stylet base 35 37 sect 03 37 sect 03 42 sect 04

(35-4) (3-4) (35-5)DGO 4 3-5 5-95 35-55 4-9 3-55 5-9

(44 sect 08 (7 sect 14 (45 sect 06 (67 sect 11 (46 sect 04 (69 sect 1

Ant end to centre of med bulb 54 45-60 41-57 45-62 46-62 48-70 42-625(522 sect 48 (518 sect 37 (532 sect 38 (56 sect 29 (58 sect 47 (555 sect 48

Length of median bulb 16 14 sect 1 117 sect 09 13 sect 1 115 sect 08 14 sect 12 115 sect 11(12-15) (95-13) (11-15) (10-13) (12-165) (10-14)

Diam of median bulb 11 95 sect 11 55 sect 05 87 sect 09 56 sect 06 10 sect 1 56 sect 05(8-12) (5-65) (7-105) (4-7) (8-12) (5-6)

Length of pharynx 81 77 sect 78 765 sect 61 757 sect 45 805 sect 45 82 sect 6 79 sect 61(64-87) (64-85) (65-86) (72-89) (72-92) (65-91)

Length of gland lobe 85 785 sect 75 646 sect 64 827 sect 71 60 sect 6 814 sect 86 625 sect 61(67-89) (52-78) (67-95) (50-74) (57-98) (55-78)

Anterior end to excret pore 86 87 sect 8 92 sect 6 856 sect 55 965 sect 53 915 sect 83 946 sect 66(73-102) (79-100) (74-97) (86-110) (79-110) (79-104)

Annule width at mid-body 12 14 sect 02 13 sect 01 13 sect 01 14 sect 01 15 sect 02 13 sect 01(11-18) (1-15) (11-17) (11-17) (12-2) (12-15)

Diam of spermatheca 10 97 sect 17 83 sect 08 10 sect 13(8-13) (7-10) (8-125)

Tail length 735 73 sect 67 765 sect 52 695 sect 8 805 sect 7 77 sect 78 815 sect 53(60-85) (65-86) (48-945) (69-93) (64-92) (73-99)

Body diam at anus 18 165 sect 19 142 sect 09 149 sect 17 145 sect 13 173 sect 3 145 sect 09(125-19) (12-16) (12-19) (125-19) (125-24) (125-16)

h 95 88 sect 09 61 sect 14 78 sect 16 59 sect 16 76 sect 12 65 sect12(8-105) (4-8) (3-11) (4-95) (45-9) (5-9)

Length of spicules 179 sect 08 175 sect 05 184 sect 06(165-19) (17-185) (17-19)

Length of gubernaculum 93 sect 05 92 sect 06 97 sect 05(85-105) (8-10) (85-11)

a 222 263 sect 34 343 sect 35 305 sect 29 37 sect 4 297 sect 38 367 sect 44(225-327) (265-40) (26-38) (295-445) (24-42) (275-45)

b 77 78 sect 05 52 sect 06 78 sect 05 61 sect 19 8 sect 05 6 sect 2(68-88) (45-78) (65-9) (48-115) (7-9) (5-12)

b0 42 43 sect 03 8 sect 08 41 sect 02 8 sect 07 45 sect 03 81 sect 08(4-5) (5-9) (37-48) (5-9) (4-55) (5-10)

554 Nematology

Radopholus duriophilus sp n from Vietnam

Table 1 (Continued)

Buon Ma Thuot Curgma Krong Ana

Holotype Paratypes

Female Female Male Female Male Female Male

c 85 83 sect 06 8 sect 04 85 sect 11 81 sect 05 87 sect 05 8 sect 04(75-95) (75-9) (6-12) (75-95) (75-10) (7-9)

c0 41 45 sect 05 55 sect 05 47 sect 07 55 sect 06 47 sect 07 56 sect 04(39-56) (45-6) (33-68) (35-65) (33-68) (5-65)

V 57 561 sect 19 564 sect 23 565 sect 19(52-59) (508-595) (538-60)

Lip region diamheight 22 27 sect 07 14 sect 02 25 sect 03 14 sect 01 25 sect 04 14 sect 01(25-5) (12-2) (2-35) (12-16) (17-3) (11-17)

Median bulb lengthdiam 15 15 sect 015 21 sect 02 15 sect 02 2 sect 03 14 sect 015 2 sect 02(12-18) (17-25) (11-19) (16-31) (12-18) (18-24)

Tail lengthstylet length 41 42 sect 03 57 sect 05 4 sect 05 6 sect 05 42 sect 04 6 sect 07(35-45) (48-65) (28-55) (5-75) (4-51) (5-73)

Width hyaline partlength 06 06 sect 01 08 sect 02 07 sect 03 07 sect 02 08 sect 01 07 sect 01(05-09) (05-16) (05-2) (04-12) (06-1) (06-1)

Tail annules 39 437 sect 55 50 sect 4 403 sect 6 515 sect 57 40 sect 32 456 sect 45(36-53) (40-58) (26-53) (39-61) (35-47) (40-61)

Fig 3 RFLP patterns of PCR products of ITS (internal tran-scribed spacer) of Radopholus duriophilus sp n M 100 bpmarker U unrestricted PCR product 1 AluI 2 RsaI 3 CfoI4 Bsp143I 5 ScrFI 6 Bsh1236I 7 Tru9I TaqI

rounded Phasmids distinct located in anterior third oftail lateral lines fusing at two thirds of distance to tailtip

Male

Slender slightly ventrally curved Cephalic region setoff knob like with three to four annules Labial discnot distinct Lateral lips terminating within third head

annule Stylet thin rudimentary with amalgamated baseMedian pharyngeal bulb oval and gland lobe poorlydeveloped Excretory pore at base of pharynx Lateral eld with four equidistant incisures at mid-body centralband of lateral eld sometimes narrower than outer bandsFour incisures at level of phasmid Oval or kidney-shaped sperm in genital tracts Postrectal intestine sacabsent Bursa leptoderan never reaching tail terminusSpicule tylenchoid with asymmetrical oval shaped headGubernaculum with head more or less prominent andpronounced pair of titillae Five to eight hypotygma onanterior cloacal aperture Tail shape conical sometimesforked terminus rounded and annulated rarely narrowand smooth

TYPE HOST AND LOCALITY

Found in association with durian (Durio zibetinusM) Buon Ma Thuot City Dak Lak province WesternHighland Vietnam

TYPE MATERIAL

One holotype nine female paratypes and six male pa-ratypes deposited in the nematode collection of the Insti-tute of Zoology Ghent University KL Ledeganckstraat35 9000 Gent Belgium six female and two male para-types and xed material from cultures deposited in the

Vol 5(4) 2003 555

CN Nguyen et al

nematode collection of the Nematology Department In-stitute of Ecology and Biological Resources 18 HoangQuoc Viet Hanoi Vietnam

DIAGNOSIS AND RELATIONSHIPS

Radopholus duriophilus sp n is characterised by thefemale cephalic region hemispherical with four to veannules female stylet length of 165-19 sup1m excretorypore located posterior to pharyngo-intestine junction inboth sexes oval or kidney-shaped sperm completelyareolated lateral eld with four incisures terminatingfar behind position of phasmid female tail conoid andtapering often with irregularly forked terminus malecephalic region knob-like with three to four annules malestylet 12-15 sup1m long male tail long and conoid hyalineportion 4-8 sup1m long bursa never reaching tail terminus

Radopholusduriophilussp n is morphologicallycloseto R similis but differs from this species by the followingdistinctive characters excretory pore located posteriorto pharyngo-intestine junction (vs at level of pharyngo-intestine junction) oval-shaped sperm (vs rod-like) fourincisures terminating far behind position of phasmid (vsthree incisures terminating at or just behind phasmid)and bursa in male never reaching tail terminus (vs bursareaching tail terminus)

Radopholus duriophilus sp n is differentiated from Rnativus by a shorter female stylet length (165-19 vs 19-23 sup1m) oval or kidney-shapedsperm (vs rod-like sperm)and four incisures at level of female phasmid (vs three) Itis further separated from R nativus by the excretory poreof both males and females being located posterior to thepharyngo-intestine junction (vs at level of or anterior topharyngo-intestine junction) and its areolated lateral eld(vs not areolated)

From R clarus Colbran 1971 R duriophilus sp n isdifferentiated by a shorter stylet length (165-19 vs 19-21 sup1m) and an areolated lateral eld (vs no areolation)From R musicola R duriophilus sp n differs by thefemale lateral eld having four equidistant incisures atmid-body (vs two deep outer folds and two faint shallowinner incisures) oval or kidney-shaped sperm (vs rod-like) rounded tail terminus (vs sharply pointed) and bymale stylet length (12-15 vs 88-12 sup1m)

From R bridgei R duriophilus sp n differs byfemale stylet length (165-19 vs 15-175 sup1m) lengthof pharyngeal median bulb (11-165 vs 11-13 sup1m)hyaline tail length (3-11 vs not more than 4 sup1m) lateral eld areolated over entire body (vs not areolated except

Table 2 Length (bp) of restriction fragments of the ITS of rDNAregions for Radopholus duriophilus sp n based on RFLP andsequence data

Enzyme Restriction fragments

AluI 377 375 144RsaI 625 248 23CfoI 393 316 176 11Bsp143I 415 298 111 72ScrFI 469 288 70 43 26Bsh1236I 621 275Tru9I 258 194 143 119 67 64 51TaqI 441 233 98 65 59

irregularly on neck and tail) male stylet length (12-15 vs10-12 sup1m) and length of hyaline tail (4-8 vs 1-4 sup1m)

MOLECULAR CHARACTERISATION

PCR ampli cation of the ITS regions of two popula-tions of R duriophilussp n yielded a single product witha length of 896 bp The ITS sequences of these two sam-ples differed in one nucleotide only All studied enzymescut the PCR products The RFLP patterns are presented inFig 3 Heterogeneity of the ITS regions was revealed byBsp143I The exact lengths of restriction fragments calcu-lated using sequence information are presented in Table 2Comparison of RFLP pro les obtained in our study withthose of R similis published by Fallas et al (1996) andElbadri et al (2002) revealed that R duriophilus sp n isdistinguished from R similis by RFLP obtained after di-gestion with Tru9I

The length of the entire ITS region for Radopholusspecies varied from 599-601 nucleotides The lengthof the alignment for Radopholus sequences was 602positions Sequence divergence ranged from 03 to 62The sequences of R duriophilus sp n differed fromthose of R similis in 28 to 37 substitutions (47-62)whereas sequences within R similis differed in two to23 substitutions (03-38) MP analysis revealed 35parsimony informative characters The single unrootedmaximum parsimonious tree is given in Fig 4 Thedistributionof R similis populationsin two main brancheson the tree is congruent with the one presented by Elbadriet al (2002) The branch with R duriophilus sp n wassupported by 24 autapomorphies (unique substitutions)

556 Nematology

Radopholus duriophilus sp n from Vietnam

Fig 4 Single unrooted parsimony tree obtained from analysis of the alignment of eight Radopholus sequences (Tree length D 56 CID 09107 HI D 00893 RI D 09020 RC D 08214) Bootstraps are given on appropriate clades

Distribution and economic importance

Out of the 96 durian trees sampled 11 were found to beinfested with R duriophilus sp n population densitiesranging from 35 to 215 individuals 5 g root and 12 to62 individuals 250 ml soil The orchards had been treatedwith carbofuran during the previous year Before thattreatment the nematode density had reached thousandsof individuals per g of root the average proportion ofdead durian trees in nurseries and recently replanted eldsbeing estimated at ca 20 In some elds up to 40of the trees had died Infected and dead trees were notonly observed in newly planted durian regions but alsoin traditional durian regions The problem however wasless important in the latter

Durian production is a speciality for Vietnam and someother countries in South East Asia In Vietnam the areaplanted to durian is 2500-3000 ha in warmer regionswith basalt soils Durian production is mainly used fordomestic consumption Indigenous cultivars have lowquality and productivity and to overcome this problemcultivars were recently imported from Thailand

Acknowledgements

This work was partly supported by the VietnameseNational Centre for Natural Sciences and Technologyand the National Fundamental Programme in NaturalSciences (Grant No 613801) SA Subbotin gratefullyacknowledges support by NATO Research FellowshipThe authors thank Mrs Rita Van Driessche Departmentof Biology Ghent University for her assistance in SEMpreparation

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Vol 5(4) 2003 557

CN Nguyen et al

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558 Nematology