articulo edgar

14
A new stem nematode associated with peanut pod rot in China: morphological and molecular characterization of Ditylenchus arachis  n. sp. (Nematoda: Anguinidae) S. L. Zhang a, G. K. Liu bc, T. Janssen c , S. S. Zhang a *, S. Xiao a , S. T. Li a , M. Couvreur c and W. Bert c a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian;  b Key Laboratory of Integrated Pest Management for Fujian    Taiwan Crops, Ministry of Agriculture, 350002 Fuzhou, Fujian, China; and  c Nematology Unit, Department of Biology, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium Sur vey s con duc ted in pea nut pro duc tion areas of Chi na rev eal ed pea nut pod rot in sev era l el ds in Sha ndo ng and Hebei Provinces, China. A large quantity of an unknown stem nematode was isolated from the hulls and seeds of pea- nuts, herein described as  Ditylenchus arachis  n. sp. The new species is characterized by a combination of the following features: lateral lip sectors distinctly projected, stylet delicate, 8 4   10  lm in length, six lines in the lateral eld, tail elongate   conoid, bursa covering about 68  86% of tail length. Pathogenicity tests showed that  D. arachis  n. sp. could infect peanut ( Arachis hypogaea), but not sweet potato ( Ipomoea batatas) or potato ( Solanum tuberosum). Morpholog- ically,  D. arachis  n. sp. appears closest to  D. africanus,  D. myceliophagus  and  D. destructor , but can be differentiated based upon a combination of morphological characteristics, host preference and molecular sequence data. The results of the phylogenetic analysis, based on 18S rDNA, the D2  D3 expansion region of 28S rDNA, and the ITS1  58S   ITS2 region, conrmed its status as a new species. A sister relationship with  D. destructor  was appointed, rather than with its ecologically very similar congener  D. africanus. Keywords:  groundnut, histopathology, molecular, morphology, pathogenicity, ribosomal DNA sequencing Introduction Pe anu t (Arach is hypo gaea), an import ant oil and food crop, is widely grown in tropical and subtropical coun- tri es, wit h a worldwid e pro duc tion est ima ted at 331 million tonnes (Fabra  et al. , 2010). Plant nematodes are the pri mar y par asit es of ground nuts in all pro duc tio n regions of the world, and are estimated to cause annual yield losses of 12% (Dickson & De Waele, 2005). Several species, such as  Meloidogyne  spp.,  Pratylenchus brachyu- rus,  Belon olaimus longicaudatus,  Cricone mella ornat a, Aphelenchoides arachidis  and  Ditylenchus africanus, are considered to be pests of great economic importance for the peanut, either worldwide or in specic regions (Shar- ma & McDonald, 1990; Dickson & De Waele, 2005). Thes e nemat ode species can attac k the roots, pegs and hulls of the peanut.  Aphelenchoides arachidis  and  D. af- ricanus  are the only two species known to invade the tes- tae of seeds.  Aphelenchoides arachidis , known as the testa nematode, only occurs on groundnuts in Nigeria (Dickson & De Waele, 2005). The peanut pod nematode,  D. afric- anus, is one of the most eco nomica lly import ant plant parasites, found in the major peanut production areas of South Africa, and it also occurs in other southern African countries (Haegeman et al., 2009). China is the world’s largest peanut producer, contribut- ing one-third of overall production (Fabra  et al. , 2010). To date, nematodes that cause severe damage to peanut in China have been restricted to the root-knot nematodes Meloidogyne hapla  and  Meloid ogyne arenaria; damage cau sed by ste m nemato des (Ditylenchus  spp .) has not been describe d yet. However, during nema tode surveys conducted in the peanut production area of China, pea- nut pod rot was found in several elds in Shandong and Hebei Provinces, and a large number of stem nematodes were iso lat ed fro m the hulls and see ds of the peanu ts. This paper describes a stem nematode species found in China infecting peanut, and herein described as  Ditylen- chus arachis  n. sp. The molecular phylogenetic afnities of  D. arachis  n. sp. with its congeneric species have been determined on the basis of rDNA sequences (18S, ITS1   58S   IT S2 , an d th e D2  D3 fr ag ment of 28 S) us in g Bayes ian infer ence and maximum likel ihood methods. Additional ly, the pathogen icity of this new stem nema- tode was assayed on peanut, potato and sweet potato. Materials and methods Stem nematode populations and morphological characterization Stem nematode-infected hull samples of peanut (cv. Xinghua no. 1) were collected in elds from four localities in two Provinces *E-mail: [email protected] These authors contributed equally to this study. ª 2013 British Society for Plant Pathology  1 Plant Pathology  (2013) Doi: 10.1111/ppa.12183

Upload: edgar-medina-gomez

Post on 13-Oct-2015

29 views

Category:

Documents


0 download

TRANSCRIPT

  • A new stem nematode associated with peanut pod rot inChina: morphological and molecular characterization ofDitylenchus arachis n. sp. (Nematoda: Anguinidae)

    S. L. Zhanga, G. K. Liubc, T. Janssenc, S. S. Zhanga*, S. Xiaoa, S. T. Lia,

    M. Couvreurc and W. Bertc

    aKey Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou,

    Fujian; bKey Laboratory of Integrated Pest Management for Fujian Taiwan Crops, Ministry of Agriculture, 350002 Fuzhou, Fujian, China;

    and cNematology Unit, Department of Biology, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium

    Surveys conducted in peanut production areas of China revealed peanut pod rot in several fields in Shandong and

    Hebei Provinces, China. A large quantity of an unknown stem nematode was isolated from the hulls and seeds of pea-

    nuts, herein described as Ditylenchus arachis n. sp. The new species is characterized by a combination of the following

    features: lateral lip sectors distinctly projected, stylet delicate, 8410 lm in length, six lines in the lateral field, tailelongateconoid, bursa covering about 6886% of tail length. Pathogenicity tests showed that D. arachis n. sp. could

    infect peanut (Arachis hypogaea), but not sweet potato (Ipomoea batatas) or potato (Solanum tuberosum). Morpholog-

    ically, D. arachis n. sp. appears closest to D. africanus, D. myceliophagus and D. destructor, but can be differentiated

    based upon a combination of morphological characteristics, host preference and molecular sequence data. The results

    of the phylogenetic analysis, based on 18S rDNA, the D2D3 expansion region of 28S rDNA, and the ITS158SITS2region, confirmed its status as a new species. A sister relationship with D. destructor was appointed, rather than with

    its ecologically very similar congener D. africanus.

    Keywords: groundnut, histopathology, molecular, morphology, pathogenicity, ribosomal DNA sequencing

    Introduction

    Peanut (Arachis hypogaea), an important oil and foodcrop, is widely grown in tropical and subtropical coun-tries, with a worldwide production estimated at 331million tonnes (Fabra et al., 2010). Plant nematodes arethe primary parasites of groundnuts in all productionregions of the world, and are estimated to cause annualyield losses of 12% (Dickson & De Waele, 2005). Severalspecies, such as Meloidogyne spp., Pratylenchus brachyu-rus, Belonolaimus longicaudatus, Criconemella ornata,Aphelenchoides arachidis and Ditylenchus africanus, areconsidered to be pests of great economic importance forthe peanut, either worldwide or in specific regions (Shar-ma & McDonald, 1990; Dickson & De Waele, 2005).These nematode species can attack the roots, pegs andhulls of the peanut. Aphelenchoides arachidis and D. af-ricanus are the only two species known to invade the tes-tae of seeds. Aphelenchoides arachidis, known as the testanematode, only occurs on groundnuts in Nigeria (Dickson& De Waele, 2005). The peanut pod nematode, D. afric-anus, is one of the most economically important plantparasites, found in the major peanut production areas ofSouth Africa, and it also occurs in other southern Africancountries (Haegeman et al., 2009).

    China is the worlds largest peanut producer, contribut-ing one-third of overall production (Fabra et al., 2010).To date, nematodes that cause severe damage to peanutin China have been restricted to the root-knot nematodesMeloidogyne hapla and Meloidogyne arenaria; damagecaused by stem nematodes (Ditylenchus spp.) has notbeen described yet. However, during nematode surveysconducted in the peanut production area of China, pea-nut pod rot was found in several fields in Shandong andHebei Provinces, and a large number of stem nematodeswere isolated from the hulls and seeds of the peanuts.This paper describes a stem nematode species found inChina infecting peanut, and herein described as Ditylen-chus arachis n. sp. The molecular phylogenetic affinitiesof D. arachis n. sp. with its congeneric species have beendetermined on the basis of rDNA sequences (18S, ITS158SITS2, and the D2D3 fragment of 28S) usingBayesian inference and maximum likelihood methods.Additionally, the pathogenicity of this new stem nema-tode was assayed on peanut, potato and sweet potato.

    Materials and methods

    Stem nematode populations and morphologicalcharacterization

    Stem nematode-infected hull samples of peanut (cv. Xinghua no.1) were collected in fields from four localities in two Provinces

    *E-mail: [email protected] authors contributed equally to this study.

    2013 British Society for Plant Pathology 1

    Plant Pathology (2013) Doi: 10.1111/ppa.12183

  • of China: population DCPXT from Julu, Xintai county; popula-

    tions DCPDC and DCPSZ from Dacui and Shangzhuan, Qianancounty, Hebei Province, respectively; and population DCPLW

    from Xinzhuang, Laiwu county, Shandong Province. Population

    DCPXT was selected as the type material. The surface of

    infected peanuts was cleaned under running tap water, while thehulls and seeds were soaked in shallow water in Petri dishes for

    24 h at room temperature. Afterwards, nematodes were col-

    lected and cultured on Alternaria longipes on potato dextroseagar (PDA) at 28C. In order to test whether other culturemedia or temperatures influence diagnostic morphometric val-

    ues, population DCPXT was also cultured on A. longipes onnutrient agar (NA) medium at 20C. After 2025 days, adultsof the four populations were collected using a modified Baer-

    mann technique. Permanent glycerol mounts were made from

    hot-formalin fixed nematodes, according to the glycerolethanolmethod (De Grisse, 1969). Measurements and drawings wereprepared manually with a camera lucida and a stage micrometer

    on an Olympus E-410 camera (Olympus Optical). Photomicro-

    graphs were taken and edited using an Olympus BH-2 micro-

    scope and PHOTOSHOP ELEMENTS v. 2.0 (Adobe). Paratype materialwas also recorded as a video clip, mimicking a multifocal obser-

    vation through a light microscope, following the video capture

    and editing procedures developed by De Ley & Bert (2002). Theresulting virtual specimens are available at http://www.nematol-

    ogy.ugent.be/vce.html.

    For scanning electron microscopy (SEM) studies, 20 males

    and 20 females were killed and fixed in 3% glutaraldehyde buf-fered with 005 M phosphate buffer (pH 68) overnight at 4C.Specimens were dehydrated in a seven-step graded series of etha-

    nol solutions, critical-point dried with liquid CO2, mounted on

    stubs with carbon discs, coated with gold (25 nm; Steel et al.,2011), viewed and photographed with a JSM-840 EM (JEOL)

    at 12 kV.

    Molecular characterization

    One female from each population was used for amplification andsequencing of the ITS158SITS2 region and D2D3 expansionregion of 28S rDNA large subunit (LSU). One female from popu-

    lation DCPXT was used for amplification and sequencing of the

    18S rDNA small subunit (SSU) region. The nematode was cutinto two pieces, transferred into PCR tubes with 8 lL distilledwater, stored at 70C for 20 min, and incubated at 99C for10 min. Then, 1 lL 10 9 PCR buffer and 1 lL proteinase K(1 mg mL1) were added to each tube, mixed and incubated at65C for 60 min, then at 95C for 10 min (Liu et al., 2011). TheDNA was used directly after extraction or stored at 20C.PCR using the universal primers was performed in a 50 lL

    reaction volume comprising 10 lL template DNA from an indi-vidual nematode, 06 lM each primer, 5 lL 10 9 PCR buffer(with Mg2+), 02 mM dNTP, 2 U rTaq DNA polymerase (TaKa-Ra Bio Inc.). The following primer pairs were used: ITSA (5-TTGATTACGTCCCTGCCCTTT-3) and ITSB (5-TTTCACTCGCCGTTACTAAGG-3) for ITS158SITS2 region (Vrainet al., 1992); D2A (5-ACAAGTACCGTGAGGGAAAGTTG-3)and D3B (5-TCGGAAGGAACCAGCTACTA-3) for the D2D3 region (Subbotin et al., 2006); G18S4 (5-GCTTGTCTCAAAGATTAAGCC-3) and 18P (5-TGATCCWKCYGCAGGTTCAC-3) for the 18S region (Blaxter et al., 1998). ThePCR reactions were performed in a MyCycler thermocycler

    (Bio-Rad) with the following conditions: 5 min at 94C; 35cycles of 1 min at 94C, 1 min at 55C and 15 min at 72C;and an additional final extension step at 72C for 10 min. PCR

    products were separated on a 1% agarose gel stained with ethi-

    dium bromide in 1 9 TAE buffer and visualized under UVlight. Cloning and sequencing of PCR products was carried out

    by Sangon Biotech (Shanghai) Co. Ltd. Three clones were

    sequenced for each product. Sequences amplified were deposited

    in GenBank. Accession numbers for the populations DCPXT,DCPDC, DCPSZ, DCPLW are as follows: JX040545,

    JN594665, JN605348, JN635037 for the ITS region; and

    JQ930029, JX145345, JX145344, JQ930028 for the D2D3region, respectively. The accession number for the 18S region of

    population DCPXT is KF219565.

    The sequenced rDNA regions were analysed with the corre-

    sponding regions of other nematodes available in GenBank. Thesequence of the ITS region of D. africanus was obtained fromGenBank (accession KF219617; Haegeman et al., 2009). Multi-ple sequence alignments of the different genes were made using

    the Q-INS-I algorithm of MAFFT v. 6.833 (Katoh & Toh, 2008)as implemented on the Bioportal server of Oslo University

    (www.bioportal.uio.no). Identical sequences were removed from

    the alignment and post-alignment trimming was done with the

    parametric profiling method of ALISCORE v. 2.2 (Misof & Misof,2009). The best-fitting substitution model was estimated using

    Akaike and Bayesian information criteria in JMODELTEST v. 2.1.2

    (Darriba et al., 2012). All genes were controlled for substitutionsaturation using the test described by Xia et al. (2003) in DAMBEv. 5.3.15 (Xia & Xie, 2001).

    Bayesian phylogenetic analysis was carried out in MRBAYES v.

    3.2.1 (Ronquist & Huelsenbeck, 2003) using the GTR + I + Gmodel. Analyses were run under default settings for 20 000 000

    generations, 25% of the converged runs were regarded as burn-

    in. Maximum likelihood analysis was conducted in RAXML v.

    7.0.4 (Stamatakis, 2006), performing 100 independent runs with1000 nonparametric bootstrap replicates under the GTRCAT

    model. Gaps were treated as missing data for all phylogenetic

    analysis. Maximum likelihood (ML) bootstrap values and pos-terior probabilities were plotted on Bayesian majority-rule con-

    sensus trees using TREEVIEW v. 1.6.6 (Page, 1996) and

    ILLUSTRATOR CS3 (Adobe). Genetic distances were calculated in

    GENEIOUS v. 6.0.5 (Biomatters; http://www.geneious.com).

    Pathogenicity assays

    The pathogenicity studies were conducted with the population

    DCPXT and a population of Ditylenchus destructor, originallyisolated from infected sweet potato from Shangdong Province.The populations were cultured on A. longipes on PDA at 28Cand nematodes were obtained using a modified Baermann tech-

    nique, centrifuged, sterilized in 01% streptomycin sulphate for5 min, washed in sterile water three times, and concentrated(5000 nematodes mL1 in water).

    Greenhouse pot experimentPeanut seeds (cv. Xinghua no. 1), sweet potato slips (cv. Qinshuno. 4) and seed potatoes with one or more eyes (cv. Bashu no. 9)

    were planted in plastic pots (15 cm diameter, 20 cm height) filled

    with steam-sterilized sandy soil (93% sand, 4% silt, 3% clay).

    Three weeks after planting, eight replicates of each plant wereinoculated with 5000 nematodes. An equal number of uninocu-

    lated plants were used as controls. The plants were irrigated with

    tap water and fertilized with compound fertilizer (65% N, 27%P, 13% K). Pots were maintained at 1830C with a 13-h photo-period. Eight weeks after inoculation, the nematodes in roots,

    pods or tubers of each plant were isolated using a modified Baer-

    mann technique.

    Plant Pathology (2013)

    2 S. L. Zhang et al.

  • Wound inoculationThe method was as described by Lin (1989). The surface of

    fresh sweet potato (cv. Qinshu no. 4) tubers was sterilized using

    75% ethanol. One hole (06 cm wide, 2 cm deep) was made bydigging out a tuber plug using a sterilized knife, then a 05 mLwater suspension containing 2500 nematodes was pipetted into

    the hole. The hole was subsequently covered with the tuber plug

    and sealed with melted wax, and the tubers were stored in anincubator at 25C. Each treatment consisted of eight replicates.Eight weeks after inoculation, the symptoms inside the sweet

    potato tuber were recorded and the nematodes were isolated

    using a modified Baermann technique.

    Histopathology

    After peanut harvest, infected testae were cut into 5-mm long

    segments, fixed in formaldehydeacetic acidethanol (FAA),dehydrated in a tertiary butyl alcohol series (40708590100%), embedded in paraffin with a melting point of 58C andsectioned with a rotary microtome. Sections (12 lm thick) werestained with safranin and fast green, mounted in a DPX medium

    (Sigma-Aldrich), examined microscopically and photographed(Vovlas et al., 2011).

    Results

    Description of peanut nematode Ditylenchus arachisn. sp.

    The measurements of holotype, 20 paratype females and20 males of population DCPXT, (Table 1) were from

    specimens cultured on A. longipes on PDA medium at28C. The main diagnostic characteristics from the otherthree populations are provided in Tables S1 (females)and S2 (males). The main measurements of males andfemales cultured on A. longipes on NA medium at 20Care also provided in Table S3.

    FemaleBody cylindrical, tapering at both ends, slightly ventralarcuate when killed by gentle heat (Fig. 1a). Cuticle withfine annulation. Head anteriorly flattened, the lip regioncontour appears smooth in two-thirds anterior and witha slight constriction annulus separated from the rest ofthe body; cephalic framework not heavily sclerotized(Figs 1b & 2a). Stoma opening pore-like, in the middleof small and circular oral disc, slightly raised, surroundedby six inner labial sensilla that open on the oral disc, sixouter labial sensilla not observed with SEM. Head-onview of labial region six-lobed in outline, the lobes corre-sponding to the hexaradiate head pattern. Subventral andsubdorsal lip sectors each with a pair of cephalic sensilla(Fig. 3a,b). Lateral lip sectors distinctly projected,extending some distance and continuous with second orthird annule, causing the first annule, or both first annuleand second annule to be discontinuous in contour, givingthe appearance of a lip region composed of four to fiveannuli. Amphidial apertures elliptical, dorsally displaced,each on the edge of lateral lip sector, situated laterallybetween first and second annule, or second and third

    Table 1 Morphometrics of females or males

    of Ditylenchus arachis n. sp. collected from

    Xintai county, Hebei Province, China. All

    measurements are in lm and in the form:

    mean SD (range)Measurement or ratio Holotype

    Paratype female

    mean SD(range)

    Paratype male

    mean SD(range)

    n 1 20 20

    L 862 893 78 (6801007) 884 91 (7301022)Lip diameter 65 70 07 (5582) 67 05 (5780)Lip height 23 24 03 (1929) 24 02 (2028)Stylet length 92 89 04 (8698) 91 04 (8596)Stylet conus length 40 39 02 (3543) 40 03 (3343)Stylet shaft length 44 44 03 (3852) 44 01 (4146)M (conus 9 100/stylet

    length)

    44 43 16 (4046) 44 22 (3948)

    Dorsal gland orifice (DGO) 10 10 01 (0913) 10 01 (0911)Body width 20 25 49 (1633) 22 30 (1828)Head to excretory pore 95 106 90 (81118) 105 57 (91115)Head to centre of

    metacorpus

    45 49 60 (3760) 49 46 (4158)

    Vulvaanus distance (VA) 109 104 15 (78130)Postvulval uterine sac (PUS) 55 57 62 (4165)Spicule length 21 22 (1624)Gubernaculum 70 12 (5090)Tail length 67 63 57 (5375) 59 38 (4963)Anal body width 12 14 25 (1120) 14 13 (1216)a 43 37 57 (2848) 41 40 (3448)b 71 71 09 (6096) 68 06 (5678)b 70 71 07 (6378) 63 09 (5273)c 13 14 12 (1116) 15 13 (1217)c 54 45 06 (3356) 44 05 (3751)V or T (%) 80 81 09 (8083) 45 86 (3665)PUS/VA (%) 50 56 75 (4374)

    Plant Pathology (2013)

    A new stem nematode on peanut 3

  • annule (Fig. 3c,d). Stylet delicate but with relativelystrong shaft, knobs relatively strong and distinctly slopingbackwards, conus comprising 4046% of total styletlength. Dorsal gland orifice (DGO) very close to styletknobs (Figs 1bd & 2a). Metacorpus (median bulb) elon-

    gate fusiform, with crescentic valves slightly anterior tocentre. Isthmus elongate, slender. Nerve ring around pos-terior part of isthmus (Fig. 1b). Hemizonid prominent,about three or four annuli long, excretory pore (EP) 81118 lm from anterior end, varying in position from

    (a) (b) (c)

    (d)

    (e)(h)

    (f) (g)

    Figure 1 Camera lucida line drawings of

    Ditylenchus arachis n. sp. (a) female entire

    body; (b, c) anterior body of female in lateral

    view; (d) female head region; (e) female tail;

    (f) male entire body; (g) anterior body of

    male in lateral view; (h) male tail.

    Plant Pathology (2013)

    4 S. L. Zhang et al.

  • opposite posterior third of isthmus to anterior third partof glandular lobe, immediately or few annuli behind thehemizonid (Fig. 2b). Basal pharyngeal bulb pyriform toquadrangular with round margins, shortly overlappingintestine (Figs 1b & 2a,b). Lateral field beginning withtwo lines at neck region (Fig. 3e), four in the anteriorbody, six in the mid-body forming five bands (Fig. 3f),four near the tail, and two in posterior end two-third ofthe tail (Fig. 3j). Ovary mono-prodelphic, outstretched,well developed. Oocytes arranged in single file (Fig. 2c).Spermatheca tubular, elongated (Figs 1a & 2d), usuallyfilled with round sperms (Fig. 2e). Uterus with prominent

    crustaformeria in form of quadricolumella of four rowsof four cells each, followed by valve-like structure anduterine sac (Figs 1a & 2d). Embryonic egg sometimespresent in uterus. Vulva close to posterior end. Vaginaperpendicular to body axis extending to half the bodywidth. Postvulval uterine sac (PUS) well developed, rela-tively broad, long, 24 02 (2126) times of vulvabody width (Fig. 2d). Anus opening arrowed (Fig. 3i).Tail about 43 times the anal body width, elongatecon-oid, usually tapering gradually to a finely to broadlyround end, slightly bent to ventral side in the posteriorend (Fig. 2f). Eggs typical for genus. Embryonic eggs

    (a)

    (f)

    (g)

    (i)

    (l)

    (m)

    (n)

    (o)

    (j)

    (k)(h)

    (b)

    (c)

    (d)

    (e)

    Figure 2 Photomicrographs of Ditylenchus

    arachis n. sp. (a) anterior body of female in

    lateral view; (b) female pharyngeal bulb,

    arrow showing excretory duct; (c) ovary

    germinal apex zone; (d) portion of female

    reproductive system and tail in ventral view,

    curly bracket showing spermatheca: (e)

    sperms in spermatheca; (f) female tail; (g)

    head of male, showing stylet and dorsal

    gland orifice; (h) anterior body of male in

    lateral view; (i) lateral lines in mid-body; (j)

    portion of male reproductive system and tail,

    showing sperms and spicule in ventral view;

    (k) spicule in lateral view; (l) tail and spicules

    in ventrallateral view; (mo) female reared

    on A. longipes on NA medium at 20C, (m,

    n) oval sperms in spermatheca; (o) egg

    bearing a juvenile in uterus. Scale bars: a, d,

    f, h, j = 25 lm; b, c, e, g, i, k, l, m, n,

    o = 10 lm.

    Plant Pathology (2013)

    A new stem nematode on peanut 5

  • (n = 20): length 56 35 (5065) lm; width 27 23(2330) lm.

    MaleMale common, male:female ratio is approximately 1:1 inall populations, i.e. isolated from hulls and seeds ofinfected peanut or reared on A. longipes on PDA. Similarto female, except for reproductive system (Fig. 1f). Headanteriorly flattened, framework weakly sclerotized, lipregion with four or five annuli, slightly narrower thanthe rest of the body (Fig. 2g). Labial area with raisedoral disc, labial region similar to female in SEM view,lateral lip sectors distinctly projected, each with a dis-tinct amphidial apertures (Fig. 3k). Stylet delicate, knobsdistinctly sloping backwards, conus comprising 3948%of total stylet length. DGO, isthmus, EP, hemizonid, asin female. Basal pharyngeal bulb pyriform, shortly over-lapping intestine (Fig. 2h). Lateral field with six lines inthe mid-body (Fig. 2i). Testis long, outstretched, roundsperms of different size (Fig. 2j). Spicules paired, arcuateventral posteriorly, weakly cephalated (Figs 1h & 2k).Gubernaculum simple, c. one-third of total spiculelength. Tail elongateconoid, straight, slightly bent toventral side in posterior part, about 43 times the analbody width, tapering gradually to a finely rounded tip.Bursa adanal, leptoderan, extending from anterior thespicula and covering about 75 42 (6886)% of thetail length (Figs 1h, 2l & 3l).

    Population DCPXT cultured on A. longipes NA med-ium at 20C showed the following differences comparedwith populations cultured on A. longipes PDA mediumat 28C: the female and male body length is relativelyshorter; spermatheca with oval sperms with distinctnucleus usually arranged in one or two rows (Fig. 2m,n)versus round sperms in vas deferens; uterus often con-tains eggs with juveniles (Fig. 2o); and bursa coveringonly about 70 49 (6279)% of tail length.

    Type host and locality

    Holotype female and additional paratypes were extractedfrom infected, discoloured peanut pods in a peanut fieldin Yan-zhuang village, Julu County, Xingtai City, HebeiProvince, China, and reared on the fungus A. longipeson PDA medium at 28C.

    Etymology

    The specific epithet arachis refers to the host plantgenus (Arachis hypogaea).

    Type specimens

    Holotype, 15 female and 10 male paratypes, mounted onglass slides were deposited in the nematology laboratorycollection (FJ1201-06) at Fujian Agriculture and Forestry

    1 m 1 m

    10 m 10 m

    10 m1 m10 m1 m

    1 m 10 m

    1 m 1 m

    (a)

    (e)

    (k) (l)

    (f)

    (h) (i)

    (g) (j)

    (b) (c) (d)

    Figure 3 Scanning electron microscope

    photographs of Ditylenchus arachis n. sp.

    (ad) female head in different views, arrows

    showing amphidial aperture; (e) anterior end,

    arrow showing beginning of lateral lines; (f)

    lateral fields in mid-body, showing six

    incisures; (g) vulva in lateral view; (h) vulva

    in ventral view; (i) anus in ventral view; (j)

    posterior end of female in lateral view, upper

    arrow showing vulva; lower arrow showing

    anus; (k) en face view of male, arrow

    showing amphidial aperture; (l) tail of male.

    Plant Pathology (2013)

    6 S. L. Zhang et al.

  • University, Fuzhou, Fujian, China. Four female and fourmale paratypes were deposited in the WANECO collec-tion, Wageningen (WT3623-25) (http://www.waneco.eu/). One female and six male paratypes were depositedat the Ghent University Zoology museum (UGMD104290-91). Voucher material is available upon requestfrom the last author.

    Differential diagnosis

    Ditylenchus arachis n. sp. is characterized by the follow-ing features: stylet 8698 lm long, conus comprisingabout 45% of the total stylet length, EP located fromthe level of the posterior one-third of the isthmus to theanterior one-third of the glandular basal bulb, six linesin the lateral field, basal bulb ventrally overlapping theintestine for a short distance, PUS c. 24 times the vulvabody width, bursa covering 6886% of the tail length,tail elongateconoid, slightly bent to the ventral side inits posterior part. In SEM, lateral lip sectors distinctlyprojected, with two amphidial apertures situated later-ally between the first and second or the second andthird annule. Its host preference is peanut. Ditylenchusarachis n. sp. also differs from related species in thesequences of the nuclear ribosomal gene cluster (seebelow).Ditylenchus arachis n. sp. is most closely related to

    D. africanus, having same preferred host and closelymorphological and molecular features, but this newspecies can be distinguished from D. africanus by itspercentage of bursa covering tail length (6886% vs4866%), position of the excretory pore (at posteriorthird of isthmus to anterior third of basal bulb versus atposterior part of basal bulb), lateral lines (6 vs 615),and relaxed body posture (slightly ventral arcuated ver-sus irregular).Ditylenchus arachis n. sp. is phylogenetically most clo-

    sely related to D. destructor but differs mainly in styletlength (8698 vs 1013 lm, respectively), spiculelength (1624 vs 2427 lm), percentage of bursa cover-ing tail length (6886% vs 5070%), posterior bulb(short, ventrally overlapping intestine versus short dor-sally overlapping intestine) and host preference (peanutversus range of host plants excluding peanut). Havingsix lines in the lateral fields and a round-ended tail,D. arachis n. sp. is also close to the Ditylenchus speciesD. caudatus, D. clarus, D. medicaginis, D. myceliopha-gus, D. triformis and D. halictus. Ditylenchus arachis n.sp. is morphologically close to D. myceliophagus butdiffers in possession of a slightly longer stylet (8698vs 6585 lm, respectively), percentage of bursa cover-ing tail length (6886% vs 2055%), and host prefer-ence (peanut versus cultivated mushroom). Ditylenchusarachis n. sp. differs from D. caudatus in possession ofa slightly shorter stylet (8698 vs 10 lm, respectively),higher percentage of bursa covering tail length (6886%vs 3550%), PUS length/vulval body diameter (>25 vs15 meta-corpus lengths posterior to the metacorpus versus at theanterior end of the isthmus to

  • Disease symptoms in the peanut field

    Visible symptoms were usually not apparent on roots orabove-ground plant material. Initiated infected pods had

    brown necrotic tissue at the point of connection withthe peg, the most distinct symptom was the develop-ment of brown to black discoloured patches, extendingacross the entire pod surface (Fig. 7a). The endocarp of

    Figure 4 The Bayesian inference 50% majority rule consensus tree generated from the 18S data set, posterior probabilities are indicated above the

    branches, bootstrap values from the maximum likelihood analysis are indicated below the branches in italics.

    Plant Pathology (2013)

    8 S. L. Zhang et al.

  • hulls of infected pods had brown or dark discolour-ation. Heavily infected seeds were shrunken and wrin-kled, testae of infected seeds were usually found withnecrotic spotty or subtle brown veins (Fig. 7b,c), the

    testae were not easily removed and the inner layer dis-played partial yellow to rust discolouration (Fig. 7d),resulting in a lower grade and reduced quantity ground-nut yield.

    Figure 5 The Bayesian inference 50% majority rule consensus tree generated from the 28S data set, posterior probabilities are indicated above the

    branches, bootstrap values from the maximum likelihood analysis are indicated below the branches in italics.

    Plant Pathology (2013)

    A new stem nematode on peanut 9

  • Pathogenicity assays

    Pot experimentThe peanut pods in eight pots inoculated with D. arachisn. sp., 8 weeks after inoculation, showed similar symp-

    toms, discoloured pegs, fewer, smaller and shrunkenpods, and discoloured endocarp of hulls or seeds(Fig. 7e,f). Thousands of D. arachis n. sp. at differentlife stages were isolated from infected groundnuts andseeds. The potato and the sweet potato tubers in all pots

    Figure 6 The Bayesian inference 50% majority rule consensus tree from generated from the ITS data set, posterior probabilities are indicated above

    the branches, bootstrap values from the maximum likelihood analysis are indicated below the branches in italics.

    Plant Pathology (2013)

    10 S. L. Zhang et al.

  • inoculated with D. arachis n. sp. or in control pots didnot show any symptoms of infection, and no nematodewas isolated from the tubers.

    Wound inoculationAfter wound inoculation of sweet potato tubers withD. arachis n. sp., no symptoms were visible and no nem-

    atodes could be isolated from the tubers. In contrast,sweet potato tubers inoculated with D. destructor werecompletely rotted (Fig. 7g), showing the typical symp-toms of dry rot including hollowness, water loss and cellshrinkage in tubers (Sun et al., 1998). Tens of thousandsof D. destructor at different life stages were isolatedfrom rotted tissues.

    (a)

    (c)

    (e)

    (h) (i)

    (j) (k)

    (f) (g)

    (d)

    (b)

    Figure 7 Symptoms caused by Ditylenchus

    arachis n. sp. on peanut. (ad) symptoms of

    infected peanut collected from field, (a)

    brown discoloured pods and pegs; (b)

    discoloured endocarp of hulls and shrunken

    seeds; (c) infected seed (right), healthy seed

    (left); (d) inner layer of the testae of seeds;

    (eg) pathogenicity assay: (e) infected

    groundnut with fewer, smaller, shrunken

    pods, discoloured peg; (f) discoloured

    endocarp of hulls and infected seeds; (g)

    sweet potato inoculated with D. arachis n.

    sp. without symptoms (left), rotted sweet

    potato inoculated with D. destructor (right);

    (hj) histopathology of infected testae: cross

    sections of parenchymatic tissues of the

    testa, showing subepidermal collapsed

    parenchyma cells surrounding the head of

    nematode, and vermiform and coiled

    nematodes in parenchyma cells (white

    arrows); (k) coiled nematodes on the outer

    layer of testa (scanning electron microscopy

    image). EP = epidermis; P = parenchyma

    tissue. Scale bars: h = 500 lm,

    ik = 100 lm.

    Plant Pathology (2013)

    A new stem nematode on peanut 11

  • Histopathology

    Ditylenchus arachis n. sp. can be found in roots, pegs,hulls and seeds, but the majority of nematodes occur inthe exocarp and endocarp of hulls, usually feeding onthe parenchyma cells and causing cellular collapse. Nem-atodes including juveniles, females, males and embryo-nated eggs were also present in the parenchyma tissue ofthe testae. Two nematode forms were found: a coiledand a vermiform nematode (arrows in Fig. 7h). Theparenchyma cells surrounding the head of vermiformnematodes were always collapsed (Fig. 7i), while theparenchyma cells surrounding the coiled nematodes didnot show distinct structural changes (Fig. 7j), and werealso found on the outer layer of testae (Fig. 7k).

    Discussion

    The genus Ditylenchus tends to be greatly conserved ingross morphology, which makes species identificationdifficult. Only a few of the morphological characteristics,the number of lines in the lateral field, stylet length, V-value, spicule length, PUS/VA (%), shape of female tailterminus, c, c, and percentage of bursa covering of taillength, are sufficiently consistent to be useful in theiridentification (Sturhan & Brzeski, 1991). In the currentstudy, these morphological characteristics maintained aconsistently stable value range in four populations ofD. arachis n. sp. collected from four localities in twoprovinces in China. Culture medium and growth temper-ature (NA medium at 20C compared with PDAmedium at 28C) considerably influenced some morpho-metrical values such as body length, but the above mor-phological characteristics remain sufficiently consistentto be used for species identification. Remarkably, spermshape and arrangement in spermatheca showed consider-able differences in the two cultures. Culture mediumand/or temperature may be responsible for the spermdevelopment, but the exact mechanism remains to beinvestigated.Amongst more than 60 species presently recognized in

    the genus Ditylenchus (Sturhan & Brzeski, 1991; Siddiqi,2000; Chizhov et al., 2010; Giblin-Davis et al., 2010;Vovlas et al., 2011), D. arachis n. sp. only shares thehost preference of peanut, with potato as a poor host,with D. africanus. Ditylenchus africanus was initiallyidentified as D. destructor based on similar morphologi-cal characteristics, and was later considered a distinctrace of D. destructor with a limited host range andpotato as a poor host (De Waele et al., 1991). Based onmorphological and molecular differences, the populationsparasitizing peanut were characterized and finally for-mally described as a new species (Wendt et al., 1995).This nematode has not been reported on groundnuts out-side of South Africa. Both D. arachis n. sp. and D. afric-anus can attack the roots, pegs, hulls and seeds of thepeanut; however, they induce some different symptomsin hulls and testae. Ditylenchus africanus induces charac-teristic symptoms with distinct dark discolouration along

    the vein that extends longitudinally along the exocarpjust beneath the pod surface, and darkened vascularstrands on the seed coat (De Waele et al., 1989),whereas D. arachis n. sp. induces the distinct symptomof brown to black discoloured patches extending alongthe entire pod surface with necrotic spotty or subtlebrown veins on the testae of infected seeds.Over recent years, phylogenetic inference has proven to

    be an effective manner of separating species of the mor-phologically conservative genus Ditylenchus (Subbotinet al., 2005; Vovlas et al., 2011). Phylogenetic analysis ofthe three ribosomal regions examined in the current studywere in agreement, and clearly separated D. arachis n.sp. from its sister species D. destructor. Based on the sin-gle available ITS sequence of D. africanus, it has beenconfirmed that D. arachis differs from D. africanus.Although the placement of these two species within theITS-based tree is not decisive, given the low bootstrapand posterior probability values, the rDNA sequence dif-ferences of D. arachis and its sister species D. destructorprovides further insight into its species status. Currentanalysis also supports the idea that the genus Ditylenchusis paraphyletic as earlier suggested by Subbotin et al.(2006).Ditylenchus arachis n. sp. is morphologically similar

    to D. destructor, a serious pest of potato production inNorth America and many parts of Europe that is alsowidely distributed throughout northern China, causingserious damage to sweet potato (Huang et al., 2010),although remarkably few reports of damage to potatohave been reported from China. The peanut, sweetpotato and potato are important crops in Shangdong andHebei Provinces, and sweet potato or potato are oftenintercropped with peanut. In surveys conducted in thepeanut production regions of these two provinces, D. de-structor has been isolated from sweet potato tubers andD. arachis n. sp. from peanut pods in different fields inthe same region of Xintai County, Hebei Province(authors unpublished data). There is a great possibilitythat D. destructor and D. arachis n. sp. could coexist inthe same field. Therefore, Ditylenchus populations iso-lated from fields where peanut production is rotated withsweet potato production should be examined morpholog-ically or using molecular characteristics to verify theidentity.The observed coiled forms in the testae indicates the

    presence of survival strategies that could enable D. ara-chis n. sp. to persist in soils or pods and overcome unfa-vourable environmental conditions (e.g. absence of ahost plant in a long dry and cold winter in northernChina). Relatively few D. arachis n. sp. individuals wereisolated from the soil or roots of peanut from infectedfields after harvest, in contrast to the high number ofnematodes obtained from hulls left in the field or storedpods (data not shown). The nematodes coiled theirvermiform bodies, most probably in response to desicca-tion as they entered into the anhydrobiotic state becausecoiling reduces the surface area of the nematode cuticlethat is exposed to the environment and thus slows drying

    Plant Pathology (2013)

    12 S. L. Zhang et al.

  • (Womersley & Higa, 1998; Treonis & Wall, 2005). Itappears that this strategy is shared with D. africanus,which can undergo complete dehydration and enter intoa state of anhydrobiosis (Basson et al., 1993). Anefficient survival stage for D. arachis n. sp. probablygenerates an important primary source of infection infields.This is the first report that a species of Ditylenchus

    can damage peanuts in China. This new species may bea potentially serious pest in peanut cultivation. Addi-tional research is needed to determine the host range,distribution and damage of this new species.

    Acknowledgements

    The authors thank A. Haegeman from ILVO (Institutefor Agricultural and Fisheries Research) for providing theITS sequence of D. africanus which was produced in theDepartment of Molecular Biotechnology, Ghent Univer-sity and D. Vlaeminck from the Department of Biology,Ghent University for histopathology technical assistance.The anonymous reviewers are acknowledged for theirdetailed and helpful comments to the manuscript. Thisstudy was supported by the National Natural ScienceFoundation of China (NFSC 31201495) and a UGentSpecial Research Fund (01NO2312).

    References

    Basson S, De Waele D, Meyer AJ, 1993. Survival of Ditylenchus

    destructor in soil, hulls and seeds of groundnut. Fundamental and

    Applied Nematology 16, 7986.

    Blaxter ML, De Ley P, Garey JR et al., 1998. A molecular evolutionary

    framework for the phylum Nematoda. Nature 392, 715.

    Chizhov VN, Borisov BA, Subbotin SA, 2010. A new stem nematode,

    Ditylenchus weischeri sp. n. (Nematoda: Tylenchida), a parasite of

    Cirsium arvense (L.) Scop. in the Central Region of the Non-Chernozem

    Zone of Russia. Russian Journal of Nematology 18, 95102.

    Darriba D, Taboada GL, Doallo R, Posada D, 2012. JMODELTEST 2:

    more models, new heuristics and parallel computing. Nature Methods

    9, 772.

    De Grisse AT, 1969. Redescription ou modifications de quelques

    techniques utilisees dans letude des nematodes phytoparasitaires.

    Mededelingen Rijksfaculteit der Landbouwwetenschappen, Gent 34,

    35169.

    De Ley P, Bert W, 2002. Video capture and editing as a tool for the

    storage, distribution, and illustration of morphological characters of

    nematodes. Journal of Nematology 34, 296302.

    De Waele D, Jones BL, Bolton C, van den Berg E, 1989. Ditylenchus

    destructor in hulls and seeds of peanut. Journal of Nematology 21,

    105.

    De Waele D, Wilken R, Lindeque JM, 1991. Response of potato

    cultivars to Ditylenchus destructor isolated from groundnut. Revue de

    Nematologie 14, 1236.

    Dickson DW, De Waele D, 2005. Nematode parasites of peanut. In: Luc

    M, Sikora RA, Bridge J, eds. Plant Parasitic Nematodes in Subtropical

    and Tropical Agriculture, 2nd edn. Wallingford, UK: CABI Publishing,

    393436.

    Fabra A, Castro S, Tauriani T et al., 2010. Interaction among Arachis

    hypogaea L. (peanut) and beneficial soil microorganisms: how much is

    it known? Critical Reviews in Microbiology 36, 17994.

    Giblin-Davis RM, Erteld C, Kanzaki N, Ye WM, Zeng YS, Center BJ,

    2010. Ditylenchus halictus n. sp. (Nematoda: Anguinidae), an

    associate of the sweat bee, Halictus sexcinctus (Halictidae), from

    Germany. Nematology 12, 891904.

    Haegeman A, Jacob J, Vanholme B, Kyndt T, Mitreva M, Gheysen G,

    2009. Expressed sequence tags of the peanut pod nematode

    Ditylenchus africanus: the first transcriptome analysis of an

    Anguinid nematode. Molecular and Biochemical Parasitology 167,

    3240.

    Huang WK, Peng DL, Zhang DS et al., 2010. Assessment of genetic

    variability in population of Ditylenchus destructor (Thorne 1945)

    (Tylenchida: Anguinidae) from China. Russian Journal of Nematology

    18, 1930.

    Katoh K, Toh H, 2008. Improved accuracy of multiple ncRNA

    alignment by incorporating structural information into a

    MAFFT-based framework. BMC Bioinformatics 9, 212.

    Lin MS, 1989. Testing the resistance of sweet-potato varieties to

    potato-rot nematode by artificial inoculation. Journal of Nanjing

    Agricultural University 12, 447.

    Liu GK, Chen J, Xiao S, Zhang SS, Pan DM, 2011. Development of

    species-specific PCR primers and sensitive detection of the Tylenchulus

    semipenetrans in China. Agricultural Sciences in China 10, 2528.

    Misof B, Misof K, 2009. A Monte Carlo approach successfully identifies

    randomness in multiple sequence alignments: a more objective means

    of data exclusion. Systematic Biology 58, 2134.

    Page RDM, 1996. TREEVIEW: an application to display phylogenetic trees

    on personal computers. Computer Applications in the Biosciences 12,

    3578.

    Ronquist F, Huelsenbeck JP, 2003. MRBAYES 3: Bayesian phylogenetic

    inference under mixed models. Bioinformatics 19, 15724.

    Sharma SB, McDonald D, 1990. A world list of plant-parasitic

    nematodes associated with groundnut. International Arachis

    Newsletter 7, 138.

    Siddiqi MR, 2000. Tylenchida Parasites of Plants and Insects, 2nd edn.

    Wallingford, UK: CABI Publishing.

    Stamatakis A, 2006. RAxML-VI-HPC: maximum likelihood-based

    phylogenetic analyses with thousands of taxa and mixed models.

    Bioinformatics 22, 268890.

    Steel H, Moens T, Scholaert A, Boshoff M, Houthoofd W, Bert W, 2011.

    Mononchoides composticola n. sp. (Nematoda: Diplogastridae)

    associated with composting processes: morphological, molecular and

    autecological characterisation. Nematology 13, 34763.

    Sturhan D, Brzeski MW, 1991. Stem and bulb nematodes, Ditylenchus

    spp. In: Nickle WR, ed. Manual of Agricultural Nematology. New

    York, USA: Marcel Dekker, Inc., 42365.

    Subbotin SA, Madami M, Krall E, Sturhan D, Moens M, 2005.

    Molecular diagnostics, taxonomy and phylogeny of the stem nematode

    Ditylenchus dipsaci species complex based on the sequences of internal

    transcribed spacer-rDNA. Phytopathology 95, 130815.

    Subbotin SA, Sturhan D, Chizhov VN, Vovlas N, Baldwin JG, 2006.

    Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2

    and D3 expansion fragments of the 28S rRNA gene sequences.

    Nematology 8, 45574.

    Subbotin SA, Deimi AM, Zheng J, Chizhov VN, 2011. Length variation

    and repetitive sequences of Internal Transcribed Spacer of ribosomal

    RNA gene, diagnostics and relationships of populations of potato rot

    nematode, Ditylenchus destructor Thorne, 1945 (Tylenchida:

    Anguinidae). Nematology 13, 77385.

    Sun JH, Peng DL, Yu KL, Bi P, Peng YK, 1998. SEM study on tissue

    pathology of stem nematode disease of sweet potato. Acta

    Agriculturae Boreali Sinica, 13, 1015.

    Treonis AM, Wall DH, 2005. Soil nematodes and desiccation survival in

    the extreme arid environment of the Antarctic dry valleys. Integrative

    and Comparative Biology 45, 74150.

    Vovlas N, Troccoli A, Palomares-Rius JE et al., 2011. Ditylenchus gigas

    n. sp. parasitizing broad bean: a new stem nematode singled out from

    the Ditylenchus dipsaci species complex using a polyphasic approach

    with molecular phylogeny. Plant Pathology 60, 76275.

    Vrain TC, Wakarchuk DA, Levesque AC, Hamilton RI, 1992.

    Intraspecific rDNA restriction fragment length polymorphism in the

    Plant Pathology (2013)

    A new stem nematode on peanut 13

  • Xiphinema americanum group. Fundamental and Applied Nematology

    15, 56373.

    Wendt KR, Swart A, Vrain TC, Webster JM, 1995. Ditylenchus

    africanus sp. n. from South Africa; a morphological and

    molecular characterization. Fundamental and Applied Nematology 18,

    24150.

    Womersley CZ, Higa LM, 1998. Trehalose: its role in the

    anhydrobiotic survival of Ditylenchus myceliophagus. Nematologica

    44, 26991.

    Xia X, Xie Z, 2001. DAMBE: software package for data analysis in

    molecular biology and evolution. Journal of Heredity 92, 3713.

    Xia X, Xie Z, Salemi M, Chen L, Wang Y, 2003. An index of

    substitution saturation and its application. Molecular Phylogenetics

    and Evolution 26, 17.

    Supporting Information

    Additional Supporting Information may be found in the online version of

    this article at the publishers web-site.

    Table S1. Main diagnostic morphometrics of females of three popula-

    tions of Ditylenchus arachis n. sp. from peanut.

    Table S2. Main diagnostic morphometrics of males of three popula-

    tions of Ditylenchus arachis n. sp. from peanut.

    Table S3. Main diagnostic morphometrics of Ditylenchus arachis n. sp.

    cultured on Alternaria longipes on nutrient agar medium at 20C.

    Plant Pathology (2013)

    14 S. L. Zhang et al.