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. Introduction

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1 Doi: /ppa 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, Fuzhou, Fujian; b Key Laboratory of Integrated Pest Management for Fujian Taiwan Crops, Ministry of Agriculture, Fuzhou, Fujian, China; and c Nematology 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 peanuts, 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, lm in length, six lines in the lateral field, 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). Morphologically, 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, 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 food crop, is widely grown in tropical and subtropical countries, with a worldwide production estimated at 331 million tonnes (Fabra et al., 2010). Plant nematodes are the primary parasites of groundnuts in all production 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 brachyurus, Belonolaimus longicaudatus, Criconemella ornata, Aphelenchoides arachidis and Ditylenchus africanus, are considered to be pests of great economic importance for the peanut, either worldwide or in specific regions (Sharma & McDonald, 1990; Dickson & De Waele, 2005). These nematode species can attack the roots, pegs and hulls of the peanut. Aphelenchoides arachidis and D. africanus are the only two species known to invade the testae of seeds. Aphelenchoides arachidis, known as the testa * shaoshengzhang@126.com These authors contributed equally to this study. Published online 16 January 2014 nematode, only occurs on groundnuts in Nigeria (Dickson & De Waele, 2005). The peanut pod nematode, D. africanus, is one of the most economically important 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, contributing 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 Meloidogyne arenaria; damage caused by stem nematodes (Ditylenchus spp.) has not been described yet. However, during nematode surveys conducted in the peanut production area of China, peanut pod rot was found in several fields in Shandong and Hebei Provinces, and a large number of stem nematodes were isolated from the hulls and seeds of the peanuts. This paper describes a stem nematode species found in China infecting peanut, and herein described as Ditylenchus arachis n. sp. The molecular phylogenetic affinities of D. arachis n. sp. with its congeneric species have been determined on the basis of rdna sequences (18S, ITS1 58S ITS2, and the D2 D3 fragment of 28S) using Bayesian inference and maximum likelihood methods. Additionally, the pathogenicity of this new stem nematode was assayed on peanut, potato and sweet potato. ª 2013 British Society for Plant Pathology 1193

2 1194 S. L. Zhang et al. Materials and methods Stem nematode populations and morphological characterization Stem nematode-infected hull samples of peanut (cv. Xinghua no. 1) were collected in fields from four localities in two Provinces of China: population DCPXT from Julu, Xintai county; populations DCPDC and DCPSZ from Dacui and Shangzhuan, Qianan county, 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 the hulls and seeds were soaked in shallow water in Petri dishes for 24 h at room temperature. Afterwards, nematodes were collected and cultured on Alternaria longipes on potato dextrose agar (PDA) at 28 C. In order to test whether other culture media or temperatures influence diagnostic morphometric values, population DCPXT was also cultured on A. longipes on nutrient agar (NA) medium at 20 C. After days, adults of the four populations were collected using a modified Baermann technique. Permanent glycerol mounts were made from hot-formalin fixed nematodes, according to the glycerol ethanol method (De Grisse, 1969). Measurements and drawings were prepared manually with a camera lucida and a stage micrometer on an Olympus E-410 camera (Olympus Optical). Photomicrographs were taken and edited using an Olympus BH-2 microscope and PHOTOSHOP ELEMENTS v. 2.0 (Adobe). Paratype material was also recorded as a video clip, mimicking a multifocal observation through a light microscope, following the video capture and editing procedures developed by De Ley & Bert (2002). The resulting virtual specimens are available at For scanning electron microscopy (SEM) studies, 20 males and 20 females were killed and fixed in 3% glutaraldehyde buffered with 005 M phosphate buffer (ph 68) overnight at 4 C. Specimens were dehydrated in a seven-step graded series of ethanol solutions, critical-point dried with liquid CO 2, 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 and sequencing of the ITS1 58S ITS2 region and D2 D3 expansion region of 28S rdna large subunit (LSU). One female from population DCPXT was used for amplification and sequencing of the 18S rdna small subunit (SSU) region. The nematode was cut into two pieces, transferred into PCR tubes with 8 ll distilled water, stored at 70 C for 20 min, and incubated at 99 C for 10 min. Then, 1 ll 109 PCR buffer and 1 ll proteinase K (1 mg ml 1 ) were added to each tube, mixed and incubated at 65 C for 60 min, then at 95 C for 10 min (Liu et al., 2011). The DNA was used directly after extraction or stored at 20 C. PCR using the universal primers was performed in a 50 ll reaction volume comprising 10 ll template DNA from an individual nematode, 06 lm each primer, 5 ll 109 PCR buffer (with Mg 2+ ), 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 -TTTCAC TCGCCGTTACTAAGG-3 ) for ITS1 58S ITS2 region (Vrain et al., 1992); D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) for the D2 D3 region (Subbotin et al., 2006); G18S4 (5 -GCTTGTC TCAAAGATTAAGCC-3 ) and 18P (5 -TGATCCWKCYGCA GGTTCAC-3 ) for the 18S region (Blaxter et al., 1998). The PCR reactions were performed in a MyCycler thermocycler (Bio-Rad) with the following conditions: 5 min at 94 C; 35 cycles of 1 min at 94 C, 1 min at 55 C and 15 min at 72 C; and an additional final extension step at 72 C for 10 min. PCR products were separated on a 1% agarose gel stained with ethidium bromide in 1 9 TAE buffer and visualized under UV light. 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, JN for the ITS region; and JQ930029, JX145345, JX145344, JQ for the D2 D3 region, respectively. The accession number for the 18S region of population DCPXT is KF The sequenced rdna regions were analysed with the corresponding regions of other nematodes available in GenBank. The sequence of the ITS region of D. africanus was obtained from GenBank (accession KF219617; Haegeman et al., 2009). Multiple sequence alignments of the different genes were made using the Q-INS-I algorithm of MAFFT v (Katoh & Toh, 2008) as implemented on the Bioportal server of Oslo University ( 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 (Darriba et al., 2012). All genes were controlled for substitution saturation using the test described by Xia et al. (2003) in DAMBE v (Xia & Xie, 2001). Bayesian phylogenetic analysis was carried out in MRBAYES v (Ronquist & Huelsenbeck, 2003) using the GTR + I + G model. Analyses were run under default settings for generations, 25% of the converged runs were regarded as burnin. Maximum likelihood analysis was conducted in RAXML v (Stamatakis, 2006), performing 100 independent runs with 1000 nonparametric bootstrap replicates under the GTRCAT model. Gaps were treated as missing data for all phylogenetic analysis. Maximum likelihood (ML) bootstrap values and posterior probabilities were plotted on Bayesian majority-rule consensus trees using TREEVIEW v (Page, 1996) and ILLUSTRATOR CS3 (Adobe). Genetic distances were calculated in GENEIOUS v (Biomatters; Pathogenicity assays The pathogenicity studies were conducted with the population DCPXT and a population of Ditylenchus destructor, originally isolated from infected sweet potato from Shangdong Province. The populations were cultured on A. longipes on PDA at 28 C and nematodes were obtained using a modified Baermann technique, centrifuged, sterilized in 01% streptomycin sulphate for 5 min, washed in sterile water three times, and concentrated (5000 nematodes ml 1 in water). Greenhouse pot experiment Peanut seeds (cv. Xinghua no. 1), sweet potato slips (cv. Qinshu no. 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 were

3 A new stem nematode on peanut 1195 inoculated with 5000 nematodes. An equal number of uninoculated 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 C with a 13-h photoperiod. Eight weeks after inoculation, the nematodes in roots, pods or tubers of each plant were isolated using a modified Baermann technique. Wound inoculation The 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 by digging out a tuber plug using a sterilized knife, then a 05 ml water 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 an incubator at 25 C. 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 formaldehyde acetic acid ethanol (FAA), dehydrated in a tertiary butyl alcohol series ( %), embedded in paraffin with a melting point of 58 C and sectioned with a rotary microtome. Sections (12 lm thick) were stained 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 arachis n. sp. The measurements of holotype, 20 paratype females and 20 males of population DCPXT, (Table 1) were from specimens cultured on A. longipes on PDA medium at 28 C. The main diagnostic characteristics from the other three populations are provided in Tables S1 (females) and S2 (males). The main measurements of males and females cultured on A. longipes on NA medium at 20 C are also provided in Table S3. Female Body cylindrical, tapering at both ends, slightly ventral arcuate when killed by gentle heat (Fig. 1a). Cuticle with fine annulation. Head anteriorly flattened, the lip region contour appears smooth in two-thirds anterior and with a slight constriction annulus separated from the rest of the body; cephalic framework not heavily sclerotized (Figs 1b & 2a). Stoma opening pore-like, in the middle of small and circular oral disc, slightly raised, surrounded by six inner labial sensilla that open on the oral disc, six outer labial sensilla not observed with SEM. Head-on view of labial region six-lobed in outline, the lobes corresponding to the hexaradiate head pattern. Subventral and subdorsal lip sectors each with a pair of cephalic sensilla (Fig. 3a,b). Lateral lip sectors distinctly projected, 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 L ( ) ( ) Lip diameter (55 82) (57 80) Lip height (19 29) (20 28) Stylet length (86 98) (85 96) Stylet conus length (35 43) (33 43) Stylet shaft length (38 52) (41 46) M (conus 9 100/stylet (40 46) (39 48) length) Dorsal gland orifice (DGO) (09 13) (09 11) Body width (16 33) (18 28) Head to excretory pore (81 118) (91 115) Head to centre of (37 60) (41 58) metacorpus Vulva anus distance (VA) (78 130) Postvulval uterine sac (PUS) (41 65) Spicule length (16 24) Gubernaculum (50 90) Tail length (53 75) (49 63) Anal body width (11 20) (12 16) a (28 48) (34 48) b (60 96) (56 78) b (63 78) (52 73) c (11 16) (12 17) c (33 56) (37 51) V or T (%) (80 83) (36 65) PUS/VA (%) (43 74)

4 1196 S. L. Zhang et al. (a) (b) (c) (f) (g) (d) (h) (e) 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. extending some distance and continuous with second or third annule, causing the first annule, or both first annule and second annule to be discontinuous in contour, giving the appearance of a lip region composed of four to five annuli. Amphidial apertures elliptical, dorsally displaced, each on the edge of lateral lip sector, situated laterally between first and second annule, or second and third annule (Fig. 3c,d). Stylet delicate but with relatively strong shaft, knobs relatively strong and distinctly sloping backwards, conus comprising 40 46% of total stylet length. Dorsal gland orifice (DGO) very close to stylet knobs (Figs 1b d & 2a). Metacorpus (median bulb) elon-

5 A new stem nematode on peanut 1197 (i) (f) (l) (a) (g) (m) 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 ventral lateral view; (m o) female reared on A. longipes on NA medium at 20 C, (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. (b) (c) (d) (e) (h) (j) (k) (n) (o) gate fusiform, with crescentic valves slightly anterior to centre. Isthmus elongate, slender. Nerve ring around posterior part of isthmus (Fig. 1b). Hemizonid prominent, about three or four annuli long, excretory pore (EP) lm from anterior end, varying in position from opposite posterior third of isthmus to anterior third part of glandular lobe, immediately or few annuli behind the hemizonid (Fig. 2b). Basal pharyngeal bulb pyriform to quadrangular with round margins, shortly overlapping intestine (Figs 1b & 2a,b). Lateral field beginning with two lines at neck region (Fig. 3e), four in the anterior body, six in the mid-body forming five bands (Fig. 3f), four near the tail, and two in posterior end two-third of the tail (Fig. 3j). Ovary mono-prodelphic, outstretched, well developed. Oocytes arranged in single file (Fig. 2c). Spermatheca tubular, elongated (Figs 1a & 2d), usually filled with round sperms (Fig. 2e). Uterus with prominent crustaformeria in form of quadricolumella of four rows of four cells each, followed by valve-like structure and uterine sac (Figs 1a & 2d). Embryonic egg sometimes present in uterus. Vulva close to posterior end. Vagina perpendicular to body axis extending to half the body width. Postvulval uterine sac (PUS) well developed, relatively broad, long, (21 26) times of vulva

6 1198 S. L. Zhang et al. (a) (b) (c) (d) 1 µm 1 µm 1 µm 1 µm (e) (f) (g) (j) (h) 10 µm 10 µm (i) (k) 1 µm (l) 10 µm 1 µm 10 µm 1 µm 10 µm Figure 3 Scanning electron microscope photographs of Ditylenchus arachis n. sp. (a d) 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. body width (Fig. 2d). Anus opening arrowed (Fig. 3i). Tail about 43 times the anal body width, elongate conoid, usually tapering gradually to a finely to broadly round end, slightly bent to ventral side in the posterior end (Fig. 2f). Eggs typical for genus. Embryonic eggs (n = 20): length (50 65) lm; width (23 30) lm. Male Male common, male:female ratio is approximately 1:1 in all populations, i.e. isolated from hulls and seeds of infected peanut or reared on A. longipes on PDA. Similar to female, except for reproductive system (Fig. 1f). Head anteriorly flattened, framework weakly sclerotized, lip region with four or five annuli, slightly narrower than the rest of the body (Fig. 2g). Labial area with raised oral disc, labial region similar to female in SEM view, lateral lip sectors distinctly projected, each with a distinct amphidial apertures (Fig. 3k). Stylet delicate, knobs distinctly sloping backwards, conus comprising 39 48% of total stylet length. DGO, isthmus, EP, hemizonid, as in female. Basal pharyngeal bulb pyriform, shortly overlapping intestine (Fig. 2h). Lateral field with six lines in the mid-body (Fig. 2i). Testis long, outstretched, round sperms of different size (Fig. 2j). Spicules paired, arcuate ventral posteriorly, weakly cephalated (Figs 1h & 2k). Gubernaculum simple, c. one-third of total spicule length. Tail elongate conoid, straight, slightly bent to ventral side in posterior part, about 43 times the anal body width, tapering gradually to a finely rounded tip. Bursa adanal, leptoderan, extending from anterior the spicula and covering about (68 86)% of the tail length (Figs 1h, 2l & 3l). Population DCPXT cultured on A. longipes NA medium at 20 C showed the following differences compared with populations cultured on A. longipes PDA medium at 28 C: the female and male body length is relatively shorter; spermatheca with oval sperms with distinct nucleus usually arranged in one or two rows (Fig. 2m,n) versus round sperms in vas deferens; uterus often contains eggs with juveniles (Fig. 2o); and bursa covering only about (62 79)% of tail length. Type host and locality Holotype female and additional paratypes were extracted from infected, discoloured peanut pods in a peanut field in Yan-zhuang village, Julu County, Xingtai City, Hebei Province, China, and reared on the fungus A. longipes on PDA medium at 28 C. Etymology The specific epithet arachis refers to the host plant genus (Arachis hypogaea).

7 A new stem nematode on peanut 1199 Type specimens Holotype, 15 female and 10 male paratypes, mounted on glass slides were deposited in the nematology laboratory collection (FJ ) at Fujian Agriculture and Forestry University, Fuzhou, Fujian, China. Four female and four male paratypes were deposited in the WANECO collection, Wageningen (WT ) ( eu/). One female and six male paratypes were deposited at the Ghent University Zoology museum (UGMD ). Voucher material is available upon request from the last author. Differential diagnosis Ditylenchus arachis n. sp. is characterized by the following features: stylet lm long, conus comprising about 45% of the total stylet length, EP located from the level of the posterior one-third of the isthmus to the anterior one-third of the glandular basal bulb, six lines in the lateral field, basal bulb ventrally overlapping the intestine for a short distance, PUS c. 24 times the vulva body width, bursa covering 68 86% of the tail length, tail elongate conoid, slightly bent to the ventral side in its posterior part. In SEM, lateral lip sectors distinctly projected, with two amphidial apertures situated laterally between the first and second or the second and third annule. Its host preference is peanut. Ditylenchus arachis n. sp. also differs from related species in the sequences of the nuclear ribosomal gene cluster (see below). Ditylenchus arachis n. sp. is most closely related to D. africanus, having same preferred host and closely morphological and molecular features, but this new species can be distinguished from D. africanus by its percentage of bursa covering tail length (68 86% vs 48 66%), position of the excretory pore (at posterior third of isthmus to anterior third of basal bulb versus at posterior part of basal bulb), lateral lines (6 vs 6 15), and relaxed body posture (slightly ventral arcuated versus irregular). Ditylenchus arachis n. sp. is phylogenetically most closely related to D. destructor but differs mainly in stylet length (86 98 vs lm, respectively), spicule length (16 24 vs lm), percentage of bursa covering tail length (68 86% vs 50 70%), posterior bulb (short, ventrally overlapping intestine versus short dorsally overlapping intestine) and host preference (peanut versus range of host plants excluding peanut). Having six lines in the lateral fields and a round-ended tail, D. arachis n. sp. is also close to the Ditylenchus species D. caudatus, D. clarus, D. medicaginis, D. myceliophagus, D. triformis and D. halictus. Ditylenchus arachis n. sp. is morphologically close to D. myceliophagus but differs in possession of a slightly longer stylet (86 98 vs lm, respectively), percentage of bursa covering tail length (68 86% vs 20 55%), and host preference (peanut versus cultivated mushroom). Ditylenchus arachis n. sp. differs from D. caudatus in possession of a slightly shorter stylet (86 98 vs 10 lm, respectively), higher percentage of bursa covering tail length (68 86% vs 35 50%), PUS length/vulval body diameter (>25 vs <1), and percentage of tail length/vulva anus distance (60% vs almost 100%). Ditylenchus arachis n. sp. differs from D. clarus in ratio a (28 48 vs 27, respectively), percentage of bursa covering tail length (68 86% vs 49 54%), the position of the nerve ring (>15 metacorpus lengths posterior to the metacorpus versus at the anterior end of the isthmus to <1 metacorpus length posterior to the metacorpus), and ovary terminus not reaching the anterior the pharyngo intestinal junction versus reaching the midpoint of the isthmus. Ditylenchus arachis n. sp. differs from D. medicaginis in slightly lower c -value (33 56 vs 4 86, respectively), percentage of bursa covering tail length (68 86% vs 20 55%), ratio of PUS/VA (43 74% vs 30 40%), and tail tip shape (rounded versus mostly pointed). It differs from D. triformis in ratio of PUS/VA (43 74% vs 25 33%, respectively), percentage of bursa covering tail length (68 86% vs 33 50%), spicule length (16 24 vs lm). Ditylenchus arachis n. sp. differs from D. halictus in its body length ( vs lm, respectively), percentage of bursa covering tail length (68 86% vs %), c -value (c = vs 50 64), SEM en face view with visible inner sensilla versus without visible cephalic sensilla, and mode of reproduction (amphimixis with a relatively even male:female ratio versus parthenogenesis with males being extremely rare), the peanut as a host plant versus the apparent lack of peanut as a host plant. Molecular and phylogenetic analysis In this study nine new sequences of D. arachis n. sp. were generated, including one 18S sequence, four D2 D3 sequences (JX and JX being identical) and four ITS sequences. After post-alignment modifications using ALISCORE, alignments were 1688 (18S), 809 (28S) and 1012 (ITS) bp long. Substitution saturation tests (Xia et al., 2003) in DAMBE indicated that alignments contained little saturation with a significant P-value for all three genes. For the different alignments, the best-fitting substitution model was estimated to be the GTR + I + G model. Phylogenetic analysis was congruent for the three rdna regions using the Bayesian and ML frameworks (Figs 4 6). In all trees, D. arachis n. sp. is placed in a moderately resolved clade as a sister group of D. destructor. Ditylenchus halictus, D. myceliophagus and D. africanus are the closest related species to this clade. Ditylenchus arachis n. sp. differs by 140 (139%) nucleotides in the ITS region in comparison to D. africanus. The rdna sequence differences between D. arachis and D. destructor are 12 15%, 91 99% and 48 67% in 18S, 28S and ITS respectively. The intraspecific variations are considerably lower, 037% (28S) and 049% (ITS) in D. arachis and 024% (18S), 17% (28S) and 20% (ITS) in D. destructor. The latter is calculated without the 191 bp hypervariable ITS consisting of repetitive elements (Subbotin et al., 2011); the intraspecific variation is 209% with this region included.

8 1200 S. L. Zhang et al. 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. Disease symptoms in the peanut field Visible symptoms were usually not apparent on roots or above-ground plant material. Initiated infected pods had brown necrotic tissue at the point of connection with the peg, the most distinct symptom was the development of brown to black discoloured patches, extending across the entire pod surface (Fig. 7a). The endocarp of hulls of infected pods had brown or dark discolouration. Heavily infected seeds were shrunken and wrinkled, testae of

9 A new stem nematode on peanut 1201 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. infected seeds were usually found with necrotic spotty or subtle brown veins (Fig. 7b,c), the testae were not easily removed and the inner layer displayed partial yellow to rust discolouration (Fig. 7d), resulting in a lower grade and reduced quantity groundnut yield. Pathogenicity assays Pot experiment The peanut pods in eight pots inoculated with D. arachis n. sp., 8 weeks after inoculation, showed similar

10 1202 S. L. Zhang et al. 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. symptoms, discoloured pegs, fewer, smaller and shrunken pods, and discoloured endocarp of hulls or seeds (Fig. 7e,f). Thousands of D. arachis n. sp. at different life stages were isolated from infected groundnuts and seeds. The potato and the sweet potato tubers in all pots inoculated with D. arachis n. sp. or in control pots did not show any symptoms of infection, and no nematode was isolated from the tubers. Wound inoculation After wound inoculation of sweet potato tubers with D. arachis n. sp., no symptoms were visible and no

11 A new stem nematode on peanut 1203 (a) (b) (c) (d) (e) (f) (g) Figure 7 Symptoms caused by Ditylenchus arachis n. sp. on peanut. (a d) 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; (e g) 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); (h j) 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, i k = 100 lm. (h) (j) (i) (k) =nematodes could be isolated from the tubers. In contrast, sweet potato tubers inoculated with D. destructor were completely rotted (Fig. 7g), showing the typical symptoms of dry rot including hollowness, water loss and cell shrinkage in tubers (Sun et al., 1998). Tens of thousands of D. destructor at different life stages were isolated from rotted tissues. Histopathology Ditylenchus arachis n. sp. can be found in roots, pegs, hulls and seeds, but the majority of nematodes occur in the exocarp and endocarp of hulls, usually feeding on the parenchyma cells and causing cellular collapse. Nematodes including juveniles, females, males and embryonated

12 1204 S. L. Zhang et al. eggs were also present in the parenchyma tissue of the testae. Two nematode forms were found: a coiled and a vermiform nematode (arrows in Fig. 7h). The parenchyma cells surrounding the head of vermiform nematodes were always collapsed (Fig. 7i), while the parenchyma cells surrounding the coiled nematodes did not show distinct structural changes (Fig. 7j), and were also found on the outer layer of testae (Fig. 7k). Discussion The genus Ditylenchus tends to be greatly conserved in gross morphology, which makes species identification difficult. 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 tail terminus, c, c, and percentage of bursa covering of tail length, are sufficiently consistent to be useful in their identification (Sturhan & Brzeski, 1991). In the current study, these morphological characteristics maintained a consistently stable value range in four populations of D. arachis n. sp. collected from four localities in two provinces in China. Culture medium and growth temperature (NA medium at 20 C compared with PDA medium at 28 C) considerably influenced some morphometrical values such as body length, but the above morphological characteristics remain sufficiently consistent to be used for species identification. Remarkably, sperm shape and arrangement in spermatheca showed considerable differences in the two cultures. Culture medium and/or temperature may be responsible for the sperm development, but the exact mechanism remains to be investigated. 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 the host preference of peanut, with potato as a poor host, with D. africanus. Ditylenchus africanus was initially identified as D. destructor based on similar morphological characteristics, and was later considered a distinct race of D. destructor with a limited host range and potato as a poor host (De Waele et al., 1991). Based on morphological and molecular differences, the populations parasitizing peanut were characterized and finally formally described as a new species (Wendt et al., 1995). This nematode has not been reported on groundnuts outside of South Africa. Both D. arachis n. sp. and D. africanus can attack the roots, pegs, hulls and seeds of the peanut; however, they induce some different symptoms in hulls and testae. Ditylenchus africanus induces characteristic symptoms with distinct dark discolouration along the vein that extends longitudinally along the exocarp just beneath the pod surface, and darkened vascular strands on the seed coat (De Waele et al., 1989), whereas D. arachis n. sp. induces the distinct symptom of brown to black discoloured patches extending along the entire pod surface with necrotic spotty or subtle brown veins on the testae of infected seeds. Over recent years, phylogenetic inference has proven to be an effective manner of separating species of the morphologically conservative genus Ditylenchus (Subbotin et al., 2005; Vovlas et al., 2011). Phylogenetic analysis of the three ribosomal regions examined in the current study were in agreement, and clearly separated D. arachis n. sp. from its sister species D. destructor. Based on the single available ITS sequence of D. africanus, it has been confirmed that D. arachis differs from D. africanus. Although the placement of these two species within the ITS-based tree is not decisive, given the low bootstrap and posterior probability values, the rdna sequence differences of D. arachis and its sister species D. destructor provides further insight into its species status. Current analysis also supports the idea that the genus Ditylenchus is 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 in North America and many parts of Europe that is also widely distributed throughout northern China, causing serious damage to sweet potato (Huang et al., 2010), although remarkably few reports of damage to potato have been reported from China. The peanut, sweet potato and potato are important crops in Shangdong and Hebei Provinces, and sweet potato or potato are often intercropped with peanut. In surveys conducted in the peanut production regions of these two provinces, D. destructor has been isolated from sweet potato tubers and D. arachis n. sp. from peanut pods in different fields in the same region of Xintai County, Hebei Province (authors unpublished data). There is a great possibility that D. destructor and D. arachis n. sp. could coexist in the same field. Therefore, Ditylenchus populations isolated from fields where peanut production is rotated with sweet potato production should be examined morphologically or using molecular characteristics to verify the identity. The observed coiled forms in the testae indicates the presence of survival strategies that could enable D. arachis n. sp. to persist in soils or pods and overcome unfavourable environmental conditions (e.g. absence of a host plant in a long dry and cold winter in northern China). Relatively few D. arachis n. sp. individuals were isolated from the soil or roots of peanut from infected fields after harvest, in contrast to the high number of nematodes obtained from hulls left in the field or stored pods (data not shown). The nematodes coiled their vermiform bodies, most probably in response to desiccation as they entered into the anhydrobiotic state because coiling reduces the surface area of the nematode cuticle that is exposed to the environment and thus slows drying (Womersley & Higa, 1998; Treonis & Wall, 2005). It appears that this strategy is shared with D. africanus, which can undergo complete dehydration and enter into a state of anhydrobiosis (Basson et al., 1993). An efficient survival stage for D. arachis n. sp. probably generates an important primary source of infection in fields.

13 A new stem nematode on peanut 1205 This is the first report that a species of Ditylenchus can damage peanuts in China. This new species may be a potentially serious pest in peanut cultivation. Additional research is needed to determine the host range, distribution and damage of this new species. Acknowledgements The authors thank A. Haegeman from ILVO (Institute for Agricultural and Fisheries Research) for providing the ITS sequence of D. africanus which was produced in the Department of Molecular Biotechnology, Ghent University and D. Vlaeminck from the Department of Biology, Ghent University for histopathology technical assistance. The anonymous reviewers are acknowledged for their detailed and helpful comments to the manuscript. This study was supported by the National Natural Science Foundation of China (NFSC ) and a UGent Special Research Fund (01NO2312). References Basson S, De Waele D, Meyer AJ, Survival of Ditylenchus destructor in soil, hulls and seeds of groundnut. 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14 1206 S. L. Zhang et al. Womersley CZ, Higa LM, Trehalose: its role in the anhydrobiotic survival of Ditylenchus myceliophagus. Nematologica 44, Xia X, Xie Z, DAMBE: software package for data analysis in molecular biology and evolution. Journal of Heredity 92, Xia X, Xie Z, Salemi M, Chen L, Wang Y, An index of substitution saturation and its application. Molecular Phylogenetics and Evolution 26, 1 7. Table S1. Main diagnostic morphometrics of females of three populations of Ditylenchus arachis n. sp. from peanut. Table S2. Main diagnostic morphometrics of males of three populations 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 20 C. Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher s web-site.