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
Surveys conducted in peanut production areas of China revealed peanut pod rot in several elds 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 eld, tail
elongateconoid, 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 ITS158SITS2
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
Peanut (Arachis hypogaea), an important oil and food
crop, is widely grown in tropical and subtropical coun-
tries, 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 brachyu-
rus, 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 specic regions (Shar-
ma & McDonald, 1990; Dickson & De Waele, 2005).
These nematode species can attack 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 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 worlds 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 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, pea-
nut pod rot was found in several elds 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 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
58SITS2, and the D2D3 fragment of 28S) using
Bayesian 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 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: shaoshengzhang@126.com

These 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, 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 col-
lected and cultured on Alternaria longipes on potato dextrose
agar (PDA) at 28C. In order to test whether other culture
media or temperatures inuence diagnostic morphometric val-
ues, population DCPXT was also cultured on A. longipes on
nutrient agar (NA) medium at 20C. After 2025 days, adults
of the four populations were collected using a modied Baer-
mann technique. Permanent glycerol mounts were made from
hot-formalin xed nematodes, according to the glycerolethanol
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). Photomicro-
graphs were taken and edited using an Olympus BH-2 micro-
scope and PHOTOSHOP ELEMENTS v. 2.0 (Adobe). Paratype material
was 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). The
resulting 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 xed 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 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 amplication and
sequencing of the ITS158SITS2 region and D2D3 expansion
region of 28S rDNA large subunit (LSU). One female from popu-
lation DCPXT was used for amplication 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 70C for 20 min, and incubated at 99C for
10 min. Then, 1 lL 10 9 PCR buffer and 1 lL proteinase K
(1 mg mL
1
) were added to each tube, mixed and incubated at
65C for 60 min, then at 95C for 10 min (Liu et al., 2011). The
DNA 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 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 ITS158SITS2 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 94C; 35
cycles of 1 min at 94C, 1 min at 55C and 15 min at 72C;
and an additional nal 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 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 amplied 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 D2D3
region, 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. The
sequence of the ITS region of D. africanus was obtained from
GenBank (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 proling method of ALISCORE v. 2.2 (Misof & Misof,
2009). The best-tting 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 substitution
saturation using the test described by Xia et al. (2003) in DAMBE
v. 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 + G
model. 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 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 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, originally
isolated from infected sweet potato from Shangdong Province.
The populations were cultured on A. longipes on PDA at 28C
and nematodes were obtained using a modied Baermann tech-
nique, 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) lled
with steam-sterilized sandy soil (93% sand, 4% silt, 3% clay).
Three weeks after planting, eight replicates of each plant were
inoculated 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 modied Baer-
mann technique.
Plant Pathology (2013)
2 S. L. Zhang et al.
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 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 modied Baermann technique.
Histopathology
After peanut harvest, infected testae were cut into 5-mm long
segments, xed in formaldehydeacetic acidethanol (FAA),
dehydrated in a tertiary butyl alcohol series (40708590
100%), embedded in parafn with a melting point of 58C 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
28C. 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 20C
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
ne annulation. Head anteriorly attened, 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 corre-
sponding 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,
extending some distance and continuous with second or
third annule, causing the rst annule, or both rst annule
and second annule to be discontinuous in contour, giving
the appearance of a lip region composed of four to ve
annuli. Amphidial apertures elliptical, dorsally displaced,
each on the edge of lateral lip sector, situated laterally
between rst 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 orice (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 relatively
strong shaft, knobs relatively strong and distinctly sloping
backwards, conus comprising 4046% of total stylet
length. Dorsal gland orice (DGO) very close to stylet
knobs (Figs 1bd & 2a). Metacorpus (median bulb) elon-
gate fusiform, with crescentic valves slightly anterior to
centre. Isthmus elongate, slender. Nerve ring around pos-
terior part of isthmus (Fig. 1b). Hemizonid prominent,
about three or four annuli long, excretory pore (EP) 81
118 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 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 eld beginning with
two lines at neck region (Fig. 3e), four in the anterior
body, six in the mid-body forming ve 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 le (Fig. 2c).
Spermatheca tubular, elongated (Figs 1a & 2d), usually
lled 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, rela-
tively broad, long, 24 02 (2126) times of vulva
body width (Fig. 2d). Anus opening arrowed (Fig. 3i).
Tail about 43 times the anal body width, elongatecon-
oid, usually tapering gradually to a nely to broadly
round end, slightly bent to ventral side in the posterior
end (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 orice; (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.
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 attened, framework weakly sclerotized, lip
region with four or ve 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 dis-
tinct amphidial apertures (Fig. 3k). Stylet delicate, knobs
distinctly sloping backwards, conus comprising 3948%
of total stylet length. DGO, isthmus, EP, hemizonid, as
in female. Basal pharyngeal bulb pyriform, shortly over-
lapping intestine (Fig. 2h). Lateral eld 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 elongateconoid, straight, slightly bent to
ventral side in posterior part, about 43 times the anal
body width, tapering gradually to a nely rounded tip.
Bursa adanal, leptoderan, extending from anterior the
spicula and covering about 75 42 (6886)% of the
tail length (Figs 1h, 2l & 3l).
Population DCPXT cultured on A. longipes NA med-
ium at 20C showed the following differences compared
with populations cultured on A. longipes PDA medium
at 28C: 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 con-
tains eggs with juveniles (Fig. 2o); and bursa covering
only about 70 49 (6279)% of tail length.
Type host and locality
Holotype female and additional paratypes were extracted
from infected, discoloured peanut pods in a peanut eld
in Yan-zhuang village, Julu County, Xingtai City, Hebei
Province, China, and reared on the fungus A. longipes
on PDA medium at 28C.
Etymology
The specic epithet arachis refers to the host plant
genus (Arachis hypogaea).
Type specimens
Holotype, 15 female and 10 male paratypes, mounted on
glass slides were deposited in the nematology laboratory
collection (FJ1201-06) at Fujian Agriculture and Forestry
1 m 1 m
10 m 10 m
10 m 1 m 10 m
1 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 elds 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 four
male paratypes were deposited in the WANECO collec-
tion, Wageningen (WT3623-25) (http://www.waneco.
eu/). One female and six male paratypes were deposited
at the Ghent University Zoology museum (UGMD
104290-91). Voucher material is available upon request
from the last author.
Differential diagnosis
Ditylenchus arachis n. sp. is characterized by the follow-
ing features: stylet 8698 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 eld, basal bulb ventrally overlapping the
intestine for a short distance, PUS c. 24 times the vulva
body width, bursa covering 6886% of the tail length,
tail elongateconoid, slightly bent to the ventral side in
its posterior part. In SEM, lateral lip sectors distinctly
projected, with two amphidial apertures situated later-
ally between the rst 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 (6886% vs
4866%), 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 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 stylet
length (8698 vs 1013 lm, respectively), spicule
length (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 (peanut
versus range of host plants excluding peanut). Having
six lines in the lateral elds and a round-ended tail,
D. arachis n. sp. is also close to the Ditylenchus species
D. caudatus, D. clarus, D. medicaginis, D. myceliopha-
gus, D. triformis and D. halictus. Ditylenchus arachis n.
sp. is morphologically close to D. myceliophagus but
differs in possession of a slightly longer stylet (8698
vs 6585 lm, respectively), percentage of bursa cover-
ing tail length (6886% vs 2055%), and host prefer-
ence (peanut versus cultivated mushroom). Ditylenchus
arachis n. sp. differs from D. caudatus in possession of
a slightly shorter stylet (8698 vs 10 lm, respectively),
higher percentage of bursa covering tail length (6886%
vs 3550%), PUS length/vulval body diameter (>25 vs
<1), and percentage of tail length/vulvaanus distance
(60% vs almost 100%). Ditylenchus arachis n. sp. dif-
fers from D. clarus in ratio a (2848 vs 27, respec-
tively), percentage of bursa covering tail length (6886%
vs 4954%), the position of the nerve ring (>15 meta-
corpus 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 pharyngointestinal junction
versus reaching the midpoint of the isthmus. Ditylenchus
arachis n. sp. differs from D. medicaginis in slightly
lower c-value (3356 vs 486, respectively), percent-
age of bursa covering tail length (6886% vs 2055%),
ratio of PUS/VA (4374% vs 3040%), and tail tip
shape (rounded versus mostly pointed). It differs from
D. triformis in ratio of PUS/VA (4374% vs 2533%,
respectively), percentage of bursa covering tail length
(6886% vs 3350%), spicule length (1624 vs 13
15 lm). Ditylenchus arachis n. sp. differs from D. halic-
tus in its body length (6801007 vs 560774 lm,
respectively), percentage of bursa covering tail length
(6886% vs 86100%), c-value (c = 3356 vs 50
64), SEM en face view with visible inner sensilla versus
without visible cephalic sensilla, and mode of reproduc-
tion (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
D2D3 sequences (JX145345 and JX145344 being iden-
tical) and four ITS sequences. After post-alignment mod-
ications using ALISCORE, alignments were 1688 (18S),
809 (28S) and 1012 (ITS) bp long. Substitution satura-
tion tests (Xia et al., 2003) in DAMBE indicated that
alignments contained little saturation with a signicant
P-value for all three genes. For the different alignments,
the best-tting 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 46). In all trees, D. arachis
n. sp. is placed in a moderately resolved clade as a sister
group of D. destructor. Ditylenchus halictus, D. myceli-
ophagus 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 1215%, 9199%
and 4867% in 18S, 28S and ITS respectively. The
intraspecic 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 con-
sisting of repetitive elements (Subbotin et al., 2011); the
intraspecic variation is 209% with this region
included.
Plant Pathology (2013)
A new stem nematode on peanut 7
Disease symptoms in the peanut eld
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 develop-
ment of brown to black discoloured patches, extending
across 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 with
necrotic 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 experiment
The peanut pods in eight pots inoculated with D. arachis
n. sp., 8 weeks after inoculation, showed similar symp-
toms, 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
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 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 nem-
atodes could be isolated from the tubers. In contrast,
sweet potato tubers inoculated with D. destructor were
completely rotted (Fig. 7g), showing the typical symp-
toms 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.
(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 eld, (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 in
the exocarp and endocarp of hulls, usually feeding on
the parenchyma cells and causing cellular collapse. Nem-
atodes including juveniles, females, males and embryo-
nated 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 identication
difcult. Only a few of the morphological characteristics,
the number of lines in the lateral eld, stylet length, V-
value, spicule length, PUS/VA (%), shape of female tail
terminus, c, c, and percentage of bursa covering of tail
length, are sufciently consistent to be useful in their
identication (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 temper-
ature (NA medium at 20C compared with PDA
medium at 28C) considerably inuenced some morpho-
metrical values such as body length, but the above mor-
phological characteristics remain sufciently consistent
to be used for species identication. Remarkably, sperm
shape and arrangement in spermatheca showed consider-
able 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
identied as D. destructor based on similar morphologi-
cal 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 nally 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 the
peanut; however, they induce some different symptoms
in hulls and testae. Ditylenchus africanus induces charac-
teristic 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 mor-
phologically 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 sin-
gle available ITS sequence of D. africanus, it has been
conrmed 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 dif-
ferences 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. de-
structor has been isolated from sweet potato tubers and
D. arachis n. sp. from peanut pods in different elds 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 eld. Therefore, Ditylenchus populations iso-
lated from elds where peanut production is rotated with
sweet potato production should be examined morpholog-
ically 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. ara-
chis n. sp. to persist in soils or pods and overcome unfa-
vourable 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
elds after harvest, in contrast to the high number of
nematodes obtained from hulls left in the eld or stored
pods (data not shown). The nematodes coiled their
vermiform bodies, most probably in response to desicca-
tion 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
Plant Pathology (2013)
12 S. L. Zhang et al.
(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
efcient survival stage for D. arachis n. sp. probably
generates an important primary source of infection in
elds.
This is the rst report that a species of Ditylenchus
can damage peanuts in China. This new species may be
a 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 (Institute
for Agricultural and Fisheries Research) for providing the
ITS sequence of D. africanus which was produced in the
Department 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 their
detailed and helpful comments to the manuscript. This
study was supported by the National Natural Science
Foundation of China (NFSC 31201495) and a UGent
Special Research Fund (01NO2312).
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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.