Nematology 00 (2012) 1-18 brill.

nl/nemy
Ditylenchus gallaeformans sp. n. (Tylenchida: Anguinidae)
a neotropical nematode with biocontrol potential
against weedy Melastomataceae
Rosngela D.L. OLIVEIRA
1,
, ngelo M. SANTIN
1
, Dalila J. SENI
1
, Anna DIETRICH
2
,
Luis A. SALAZAR
3
, Sergei A. SUBBOTIN
4,5
, Manuel MUNDO-OCAMPO
6
,
Renato GOLDENBERG
7
and Robert W. BARRETO
1
1
Departamento de Fitopatologia, Universidade Federal de Viosa, Viosa, MG, 36570-000, Brazil
2
Leibniz Universitt Hannover, Institut fr Panzenkrankheiten, Herrenhuser Strae 2, D-30419 Hannover, Germany
3
Laboratorio de Nematologia, Faculdad de Agronomia, Universidad de Costa Rica, San Jose, Costa Rica
4
Plant Pest Diagnostics Center, California Department of Food and Agriculture,
3294 Meadowview Road, Sacramento, CA 95832-1448, USA
5
Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences,
Leninskii Prospect 33, Moscow, 117071, Russia
6
Nematologia CIIDIR-IPN, Unidad Sinaloa, Mexico
7
Departamento de Botnica, Universidade Federal do Paran, Cx.P. 19031, Curitiba, PR 81531-70, Brazil
Received: 22 February 2012; revised: 1 June 2012
Accepted for publication: 13 June 2012
Summary Ditylenchus gallaeformans sp. n. was found on several hosts at numerous locations in Brazil and Costa Rica. In its
native habitats it attacks several genera in the Melastomataceae, including two species ranked as among the worst invasive weeds
of Pacic island forests, namely Miconia calvescens and Clidemia hirta. The new species causes a severe disease on infected plants
involving the formation of gall-like structures on infected leaves, inorescences and stems, and may cause signicant impact on its
hosts. Morphological study using light and scanning electron microscopy and analysis of the partial 18S rRNA, the D2-D3 expansion
fragments of 28S rRNA and the ITS rRNA gene sequences showed little variations between populations from different hosts or
geographical origins. The molecular study revealed that the new species is related to D. drepanocercus, which was recently found
in association with M. calvescens but causing angular leaf spots on this host. Ditylenchus gallaeformans sp. n. is distinguished from D.
drepanocercus by having a bursa reaching the tail tip (vs covering around 50% of tail in D. drepanocercus) and a conical tail, regularly
tapering towards a variable tip (vs tail with a distinctive apical falciform appendage in both sexes in D. drepanocercus). PCR with
species-specic primers was developed for diagnostics of both Ditylenchus species. Ditylenchus gallaeformans sp. n. deserves further
investigation as a potential biocontrol agent against M. calvescens and C. hirta.
Keywords biological control, Brazil, Clidemia hirta, Costa Rica, description, Ditylenchus drepanocercus, Miconia calvescens,
molecular, morphology, morphometrics, phylogeny, taxonomy.
Three neotropical plant species in the Melastomat-
aceae are presently of special relevance as invaders in
the Pacic, namely: Clidemia hirta (L.) D.Don, Mico-
nia calvescens DC. and Tibouchina herbacea (DC.) Cogn.
(Cronk & Fuller, 1995; DeWalt et al., 2004). Miconia
calvescens is the worst weed among these ecosystem in-
vaders. Pacic island native forest ecosystems, particu-

Corresponding author, e-mail: [email protected]

larly those of French Polynesia, the Hawaiian Islands and
Australia, have been experiencing invasions by this pre-
viously obscure shrub or small tree species since its in-
troduction in the mid-20th century (Medeiros et al., 1997;
Meyer, 1998). Mechanical removal and chemical control
have been successful in some parts of Hawaii in prevent-
ing its expansion into new areas, but a more complete and
Koninklijke Brill NV, Leiden, 2012 DOI:10.1163/15685411-00002670
R.D.L. Oliveira et al.
permanent control is likely to be reached only through the
classical introduction of biocontrol agents collected in the
centre of origin (Medeiros et al., 1997).
In 1995 a cooperative work involving Brazilian and
American organisations was started, aimed at collect-
ing and investigating potential biocontrol agents of M.
calvescens and other invasive Melastomataceae from
Brazil and the neotropical region. The potential biocontrol
agent list included: a phytoplasma (Seixas et al., 2002)
causing witches broom; a plant-parasitic nematode, Dity-
lenchus drepanocercus Goodey, 1953, causing angular
leaf spots (Seixas et al., 2004a); an oomycete, Pythium
sp., causing root rot; as well as numerous fungi (Seixas et
al., 2007; Alves et al., 2010) and some insect species col-
lected in Brazil (Picano et al., 2005; Burckhardt et al.,
2006) and Costa Rica (Buckhardt et al., 2005). Until re-
cently, only one natural enemy, the fungus Colletotrichum
gloeosporioides f. sp. miconiae, has been released for
classical biological control of M. calvescens, being intro-
duced into the Hawaiian Islands in 1997 (Killgore et al.,
1999) and French Polynesia in 2001 (Meyer et al., 2008).
In June 2004, while examining plant abnormalities and
galls supposedly caused by eriophyid mites in Miconia
spp. in Costa Rica and Mexico, an entomologist, Alec
McClay (McClay Ecoscience), discovered several nema-
tode specimens associated with dissected galls. Further
detailed examination of plant samples revealed that ne-
matode galls were also rather common in Brazil on vari-
ous species of Miconia and other Melastomataceae. The
nematode specimens were identied as representatives of
the genus Ditylenchus Filipjev, 1936, but they seemed
to be different from D. drepanocercus, which was previ-
ously recorded as attacking M. calvescens in the neotrop-
ics (Seixas et al., 2004a, b) and causing a different kind of
symptom (angular leaf spots). This publication includes a
description of a new species of Ditylenchus found in as-
sociation with M. calvescens. Only systematic aspects are
presented in this publication: other aspects, including life-
cycle, gall development, impact on host plant, host range
and epidemiology are in preparation and will be published
separately.
Materials and methods
NEMATODE POPULATIONS AND SAMPLING
Leaves and inorescences infected with nematodes
were collected from plants at an easily accessible location
in the vicinity of the campus of the Federal University
of Viosa (Chcara Cristais, municipality of Viosa, state
of Minas Gerais, Brazil). Specimens belonging to other
populations were also collected from members of the
Melastomatacae in several municipalities of Minas Gerais
State (MG) and from one site in Costa Rica. Some
samples were submitted to morphometric and molecular
analyses (Table 1). Although galls and foliage distortions
caused by Ditylenchus were observed on M. calvescens
in Brazil, another species of Miconia, M. ibaguensis
(Bonpl.) Triana, was found to be infected by even larger
populations of this nematode. This species became a
convenient source of nematodes throughout the year and
for collecting the material used in the description of the
new species and in inoculation experiments. Additionally,
D. drepanocercus was collected again from M. calvescens
in Brazil and compared with the new species.
NEMATODE EXTRACTION, FIXATION AND MOUNTING
Galled tissues were separated from leaves, cut into
small pieces and placed in a 250 ml ask lled with tap
water. The material was kept for 24-48 h under continuous
aeration provided by an aquarium pump. The resulting
suspension was poured through an 840 m pore sieve
to remove leaf debris and through a 37 m pore sieve
to extract the nematodes from the suspension. Nematodes
were killed in a water bath (55C for 4 min) and xed in
TAF (triethanolamine formaldehyde) for 48 h. Nematode
specimens were mounted in glycerin on permanent slides
by following Seinhorsts (1959) method.
LIGHT AND SCANNING ELECTRON MICROSCOPY
Nematode specimens were studied with an Olympus
BX51 equipped with a Nomarski differential interference
contrast and a drawing tube. Light photographs of females
and males were taken with a digital camera (Olympus
E-volt 330) attached to the microscope.
For SEM observations, adult males and females were
obtained from infected M. ibaguensis. Specimens were
heat-relaxed with hot water (65C) and immediately
xed with a 5% buffered (pH 7.0) formalin solution for
24 h. After primary xation in a 5% formalin solution
nematodes were rinsed with several changes of 0.1 M
phosphate buffer and then transferred to a beam capsule,
followed by a post-xation period of 4 h in a 4%
OsO
4
solution. Post-xed specimens were rinsed with
several changes of cold 0.1 M phosphate buffer within
a 15 min period and then dehydrated through a series
of absolute ethanol aqueous dilutions from 20% through
2 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
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Vol. 00(0), 2012 3
R.D.L. Oliveira et al.
100%. Dehydrated specimens were critical point-dried in
a Tousimis autosamdri-810 critical point drier, mounted
on aluminium stabs, coated with a 25 nm layer of gold
palladium and observed with a XL30 Phillips SEM at
10 Kv.
DNA EXTRACTION, PCR, CLONING AND
SEQUENCING
Several nematode individuals from sampled popula-
tions (Table 1) were transferred to a series of separate
Eppendorf tubes, each containing 16 l ddH
2
O, 2 l
10 PCR buffer and 2 l Proteinase K (600 g ml
1
)
(Promega) and crushed with an ultrasonic homogeniser.
The tubes were incubated at 65C (1 h) and then at 95C
(15 min). After incubation, the tubes were centrifuged
for 2 min at 15 000 g and kept at 20C until use.
Detailed protocols for PCR, cloning and automated se-
quencing were as described in Tanha Maa et al. (2003).
The 18S rRNA gene was amplied using two sets of
primers (two overlapping fragments): i) forward G18SU
(5

-GCTTGTCTCAAAGATTAAGCC-3

) (Blaxter et al.,
1998) and reverse R18Tyl1 (5

-GGTCCAAGAATTTCA
CCTCTC-3

) (Chizhov et al., 2006); and ii) forward

F18Tyl2 (5

-CAGCCGCGGTAATTCCAGC-3

) and re-
verse R18Tyl2 (5

-CGGTGTGTACAAAGGGCAGG-3

)
(Chizhov et al., 2006). The ITS1-5.8S-ITS2 rRNA gene
region was amplied using a set of primers: forward
TW81 (5

-GTTTCCGTAGGTGAACCTGC-3

) and re-
verse AB28 (5

-ATATGCTTAAGTTCAGCGGGT-3

)
(Subbotin et al., 2005). For several samples, only the ITS1
rRNA gene region was amplied with forward TW81
and reverse 5.8SM5 (5

-GGCGCAATGTGCATTCGA-3

)
primers as described by Zheng et al. (2000). The D2-D3
expansion segments of 28S rRNA gene were amplied
using forward D2TylB (5

-GAGAGAGTTAAANAGBAC
GTG-3

) and reverse D3B (5

-TCGGAAGGAACCAGCT
ACTA-3

) primers. The newly obtained sequences were

deposited in GenBank under the accession numbers indi-
cated in Table 1.
PCR WITH SPECIES-SPECIFIC PRIMERS
Several Ditylenchus samples were used to test two
species-specic primers. The PCR mixture was prepared
as described in Tanha Maa et al. (2003). The universal
forward TW81 primer was used in PCR with combina-
tions of the specic reverse D_drep (5

-TCAGCCAAGCC
AGACAAGTCAGT-3

) or the specic reverse D_gall (5

-
TGGCACACTCTTGGACTGATGCT-3

) primers for di-

agnosis of D. drepanocercus or D. gallaeformans sp. n.,
respectively. The PCR amplication prole consisted of
4 min at 94C; 30 cycles of 1 min at 94C, 45 s at 57C
and 45 s at 72C, followed by a nal step of 10 min at
72C. Four l of the PCR product was run on a 1.4% TAE
buffered agarose gel, stained and photographed.
SEQUENCE AND PHYLOGENETIC ANALYSIS
The newly obtained sequences for 18S rRNA and D2-
D3 of 28S rRNA genes were aligned using ClustalX 1.83
with default parameters with corresponding published
gene sequences of Anguinidae and several outgroups
(Holterman et al., 2006; Subbotin et al., 2006; Meldal
et al., 2007; Davies et al., 2009; Zhao et al., 2011),
respectively. The best t model of DNA evolution was
obtained using the program MrModeltest 2.2 (Nylander,
2002) with the Akaike Information Criterion in conjunc-
tion with PAUP*. Sequence datasets were analysed with
Bayesian inference (BI) using MrBayes 3.1.2 (Huelsen-
beck & Ronquist, 2001) with the general time-reversible
(GTR) model of nucleotide substitution including a pro-
portion of invariable sites (I) and a gamma distribution (G)
of among-site-rate heterogeneity with six rate categories.
The analysis was initiated with random starting trees and
was run with four chains for 1.0 10
6
generations. The
Markov chains were sampled at intervals of 100 gener-
ations. After discarding burn-in samples and evaluating
convergence, the remaining samples were retained for fur-
ther analysis. The topologies were used to generate a 50%
majority rule consensus tree.
Results
SYMPTOMS OF INFECTION
Upon closer examination, symptoms caused by erio-
phyid mite attack, although involving foliage distortions
as in infection by the novel nematode species, were recog-
nised as being clearly distinct. Distorted tissues were al-
ways blister-like and accompanied by velvety erynose.
Larviform eriophyid mites were always found within the
erynose but attempts to extract nematodes from such
tissues yielded no Ditylenchus individuals. Symptoms
caused by the new gall-forming Ditylenchus were similar
for all host plant species. Nematodes induced severe fo-
liage and inorescence distortions mostly associated with
gall-like formation of various sizes (Fig. 1B-F). On leaves,
symptoms were amphigenous on the lamina and were also
4 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
Fig. 1. Field symptoms. A: Intense velvety erynose and pronounced blister-like leaf distortion on Miconia ibaguensis caused by an
unidentied eriophyid mite (nematodes absent from erynose); B: Abundant galls on inorescences and leaves on M. ibaguensis caused
by Ditylenchus gallaeformans sp. n.; C, D: Foliar deformation resulting from parasitism of D. gallaeformans sp. n. on M. calvescens
(on C note miniature upward oriented leaves (arrowed) and detail of gall (inset); on D note drastic deformation of leaf due to nematode
attack rendering it cabbage-shaped); E: Healthy branch of M. corallina (left) besides a branch overrun by abundant leaf-galling (right);
F: Clidemia hirta individual plant-bearing galls and foliage distortion as the result of D. gallaeformans sp. n. infection.
Vol. 00(0), 2012 5
R.D.L. Oliveira et al.
seen on veins and petioles close to the stems. Complete
distortion of the apical bud, followed by the formation of
groups of galls or larger galls up to 5 cm diam., was also
common. Additionally, few to numerous miniature leaves
arising vertically from infected areas of the lamina were
often observed (Fig. 1C). Infection of inorescences led
to the formation of galls instead of fruits. On some hosts,
such as Clidemia capitellata (Bonpl.) D.Don and Miconia
latecrenata (DC.) Naudin, galls on stems were also com-
monly observed and sometimes were even more abundant
than on other organs. No evidence of immediate necro-
sis was seen on gall tissue and they appeared to become
gradually senescent together with neighbouring healthy
tissue. Articial inoculation studies (data not shown) in-
dicated that there is a latent period of approximately 30
days from inoculation to onset of gall formation. Galls
emerged mostly next to leaf veins and were always on
expanding young leaves. Infection points appeared rstly
as small pale dots representing areas where dense forma-
tions of hyaline trichomes were formed. Galls developed
from these points and increased gradually in size. Galls
were usually formed by the disorganised growth of young
foliar tissues leading to the formation of galleries and cav-
ities where numerous trichomes were formed and the ne-
matodes concentrated. After repeated and detailed micro-
scopic examinations of plant sections of infected tissues
of different hosts and different pathogenesis stages, no
evidence was found of the presence of nematodes inside
the plant tissues. Nematode individuals were only found
abundantly in the deep crevices and open-ended crypts of
galls. A preliminary report of a study of the gall devel-
opment was recently published (Santin et al., 2009) and
a histopathological analysis will be published separately
in detail showing that this species is an ectoparasite of
melastomes forming populations that congregate on galls.
Ditylenchus gallaeformans
*
sp. n.
= Ditylenchus gallaeformans apud
Santin et al., 2009
(Figs 2-5)
MEASUREMENTS
See Tables 2 and 3.
*
Specic epithet referring to the galls formed by this nematode
on infected hosts.
DESCRIPTION
Female
Body posture, after xation and mounting in glycerin,
straight to slightly arcuate. Cuticular annulation and lat-
eral eld weakly expressed, annuli 1-2 m wide; lateral
eld with four lines. Under light microscope, four lines
almost indistinct, seemingly equidistant. Lateral eld only
observed in a few females and males, lines as faint as
transverse annuli. Head low, not offset, 5.1 m broad
and 1.7 m high, framework not sclerotised. Head annuli
indistinct under LM. Under SEM, face view with quad-
rangular outline. Stylet delicate, knobs rounded, well de-
veloped, 1.5-2.6 m wide, conical part about half stylet
length. Pharynx with subcylindrical procorpus, median
bulb oval, 51 2.2 (47-55) m from anterior end, with
central sclerotised thickenings of lumen. Isthmus narrow,
cylindrical, 1.5-1.8 times as long as procorpus. Basal bulb
pyriform, offset, sometimes slightly overlapping intestine,
large dorsal nucleus often distinct, subventral gland nu-
clei not seen. Nerve ring surrounding isthmus, hemizonid
located from excretory pore level to 3 m anteriorly. Ex-
cretory pore 97 11.8 (67-109) m from anterior end.
Female reproductive system with characteristics of Dity-
lenchus, reproductive tract prodelphic, ovary outstretched,
oocytes mainly in single row, uterine quadricolumellar
followed by an elongated spermatheca packed with large
rounded sperm, vulva a transverse slit, vagina somewhat
oblique to body axis, reaching more than halfway across
body. Post-uterine sac covering almost twice (1.4-1.8)
vulval body diam. and extending one-fth to one-seventh
of vulva-anus distance (PUS%VA). Eggs (paratypes; n =
50) 54 5.3 (49-61) 24 1.3 (18-25) m in size.
Tail conical, regularly tapering towards a variable tip, fre-
quently regularly pointed to minutely rounded, sometimes
exhibiting annulations at posterior end.
Male
Similar to female but usually shorter. Spicules short,
simple, curved ventrally, gubernaculum simple, short.
Testis usually reexed at anterior end. In lateral view,
bursa totally covering tail, beginning before proximal end
of spicule and extending to tail tip.
Second-stage juveniles
Second-stage juvenile paratypes (n = 20) with follow-
ing dimensions: L = 215 25.2 (184-295) m; stylet
length = 7.2 0.5 (6.4-7.8) m; tail length = 20.7 2.5
(16.0-25.6) m.
6 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
Fig. 2. Ditylenchus gallaeformans sp. n. A: Entire female body; B: Anterior end of female; C: Entire male body; D: Posterior end of
male; E: Posterior ends of (from left to right): one male and two females; F: Posterior female end; G: Female body at vulva level. (Scale
bars: A, C, E-G = 50 m; B = 10 m; D = 20 m.)
Vol. 00(0), 2012 7
R.D.L. Oliveira et al.
Fig. 3. Light photomicrographs of Ditylenchus gallaeformans sp. n. A: Entire female; B: Anterior end of female; C: Posterior end of
female; D: Vulval region with post-uterine sac; E: Posterior end of male. (Scale bars: A = 25 m; B-D = 10 m; E = 20 m.)
8 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
Fig. 4. Scanning electron photomicrographs of Ditylenchus gallaeformans sp. n. A: Sublateral view of female showing head annulation;
B: Face view showing labial plate and position of amphids (arrow); C: Lateral view showing lateral eld at vulval region; D: Tail of a
female showing end of lateral eld; E: Lateral view of male showing bursa and protruding spicule; F: Ventral view of tail.
Vol. 00(0), 2012 9
R.D.L. Oliveira et al.
Fig. 5. Sequence alignment of the ITS1-5.8S-ITS2 rRNA gene for Ditylenchus gallaeformans sp. n. from Miconia corallina and D.
drepanocercus. The 18S, 5.8S and 28S rRNAgene sequences are marked in bold. The positions of three universal primers are underlined
and the position of two species-specic primers are underlined and marked by grey in the sequence alignment.
10 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
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Vol. 00(0), 2012 11
R.D.L. Oliveira et al.
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12 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
NOTE
Biometric data for either sex did not vary signicantly
between different populations collected from various
hosts and geographic origins (Tables 2, 3). The largest
females and males were found in Leandra lacunosa and
the smallest in M. albicans. For these samples, ratio a
ranged from 26 to 34, but other character variations were
minimal. The vulval position was consistent (V = 72-
75). Additionally, a nematode population obtained from
M. mendoncae had a larger PUS%VA (19.4) compared
to other samples. None of these differences can be
considered as sufcient to justify separate taxonomic
status for any sample.
This publication formerly proposes a binomen which
was previously mentioned in the literature in a conference
abstract (Santin et al., 2009) but without a complete and
formal description.
TYPE HOST AND LOCALITY
Miconia ibaguensis (Bonpl.) Triana leaves and inores-
cences collected in Cristais, Viosa, Minas Gerais State,
Brazil, by R.W. Barreto (RWB739) on 16 December 2004.
OTHER HOSTS AND LOCALITIES
Initially, six species of Miconia, C. capitellata, C. hirta
and Leandra lacunosa Cogn. were found to be infected
with the nematode in the state of Minas Gerais, Brazil.
Nematode infection was reported on M. calvescens from
Costa Rica (Turrialba) (Table 1). Later, when the survey
was expanded, numerous additional geographical and host
plant records of this species were recorded in Brazil
(Oliveira et al., unpubl.) and Costa Rica (Dietrich, 2006).
TYPE MATERIAL
Holotype, female and male paratypes, mounted on
glass slides deposited at the United States Department of
Agriculture Nematode Collection (accession numbers: T-
6132-T-6138; seven slides, each with a male and female),
Beltsville, MD, USA, and University of California River-
side Nematode Collection (30722-30727; six slides, each
with a male and female), Riverside, CA, USA.
DIAGNOSIS AND RELATIONSHIPS
Ditylenchus gallaeformans sp. n. is characterised by
straight to slightly arcuate body 442-698 m in length, lip
region not offset, presence of four incisures, slender stylet
6.4-9.0 m in length, basal bulb slightly overlapping
the intestine, post-uterine sac 1.4-1.8 vulval body diam.
long and conical tail with regularly pointed to minutely
rounded terminus.
Ditylenchus gallaeformans sp. n. is most similar to D.
drepanocercus, a species previously described in associ-
ation with angular leaf spot on M. calvescens (Seixas et
al., 2004a). The latter species has a distinctive falciform
appendage on the apex of the tail in both sexes, which al-
lows for its easy separation from D. gallaeformans sp. n.
Additionally, the male bursa of D. drepanocercus covers
50% of tail whereas in D. gallaeformans sp. n. it covers
the entire tail (Brzeski, 1991; Seixas et al., 2004a).
Ditylenchus gallaeformans sp. n. differs from other
plant-parasitic Ditylenchus (D. angustus (Butler, 1913)
Filipjev, 1936, D. dipsaci (Khn) Filipjev, 1936 and D.
dryadis Anderson & Mulvey, 1980) as follows: from D.
angustus by having a shorter body and a shorter stylet
(L =0.6 (0.59-0.64) vs 0.8-1.2 mm; stylet =7.7 (6.5-8.3)
vs 10-11 m) and also by the absence of a mucronate tail
vs mucronate in D. angustus; from D. dipsaci by having
a shorter body and stylet (L = 0.6 (0.59-0.64) vs 1.0-
1.3 mm; stylet = 7.7 (6.5-8.3) vs 10-12 m), and lower
values for V and PUS%VA (V = 74.6 (72.7-77.3) vs
76-86; PUS%VA = 16.7 (12.7-19.0) vs 40-70); from D.
dryadis by having four vs 6-8 incisures in the lateral eld,
and lower V and PUS%VA (V = 74.6 (72.7-77.3) vs 80-
83; PUS%VA = 16.7 (12.7-19.0) vs 61-86). Additionally,
D. gallaeformans sp. n. has the bursa extending over
the entire tail whereas it extends to less than 100% in
D. angustus, and covers 40-70% of the tail length in D.
dipsaci and 64-76% in D. dryadis.
Although the presence of a bursa covering the entire
tail in D. gallaeformans sp. n. is a very distinctive
feature, it is not exclusive to this species. There are four
other species of Ditylenchus having males with a bursa
extending over almost the whole length of tail, namely:
D. cyperi Husain & Khan, 1967, D. nanus Siddiqi, 1963,
D. mirus Siddiqi, 1963 and D. virtudesae Tobar-Jimenez,
1964 (Brzeski, 1991; Siddiqi, 2000). The new species can
be distinguished from D. cyperi by four vs ve incisures in
the lateral eld, higher ratio a (31.2 (25.0-35.7) vs 18-29
in females of D. cyperi) and lower PUS%VA (16.7 (12.7-
19.0) vs 50); from D. nanus by four vs six incisures, lower
PUS%VA (16.7 (12.7-19.0) vs 60-70) and longer spicules
(18.6 (16.6-25.6) vs 14-15 m); from D. mirus by having
a pointed to slightly rounded tail tip vs broadly rounded
and lower V value of 74.6 (72.7-77.3) vs 83-85; and from
D. virtudesae by number of four vs six incisures, a pointed
Vol. 00(0), 2012 13
R.D.L. Oliveira et al.
to slightly rounded tail tip vs broadly rounded and lower
V value of 74.6 (72.7-77.3) vs 79.9-81.7.
MOLECULAR CHARACTERISATION AND DIAGNOSTICS
OF Ditylenchus gallaeformans SP. N. AND
D. drepanocercus
Sizes of PCR products amplied by TW81 and AB28
primers were ca 946-948 and 895 bp for D. gallaeformans
sp. n. and D. drepanocercus, respectively. The sequence
alignment showing the differences in the ITS rRNAregion
between these species is given in Figure 5. Ten obtained
sequences from different populations and PCR cloned
products of D. gallaeformans sp. n. differed from each
other by three nucleotides and two insertions/deletions
(TG in ITS1 and TTTA in ITS2) only. Comparison of
ITS rRNA gene sequences for D. gallaeformans and D.
drepanocercus with those belonging to the Ditylenchus
dipsaci species complex (Subbotin et al., 2005) revealed
a substantial divergence between these groups (data not
shown).
In our study, we designed specic primers for both
Ditylenchus species. The results of PCR with these
primers are given in Figure 6. The combination of the
universal TW81 primer with the species-specic D_gall
primer yielded an amplicon of ca 173 bp in length for
all D. gallaeformans sp. n. samples and the combination
of the universal TW81 primer with the species-specic
D_drep primer yielded an amplicon of 313 bp in length
for a D. drepanocercus sample (Fig. 6).
PHYLOGENETIC RELATIONSHIPS OF Ditylenchus
gallaeformans SP. N. WITH OTHER ANGUINIDAE
The alignment of partly sequenced 18S rRNA gene was
709 bp in length and contained 19 taxa including three
outgroup taxa. Phylogenetics analysis of the 18S rRNA
gene sequences using BI revealed close relationships of
D. gallaeformans sp. n. and D. drepanocercus. These
Ditylenchus species formed a highly supported clade
(posterior probability = 99) in the majority consensus BI
tree (Fig. 7).
The alignment of D2-D3 expansion segments of 28S
rRNA gene was 695 bp in length and contained 18
taxa, including two outgroup taxa. The phylogenetic
relationships of Ditylenchus with other Anguinidae is
presented in Figure 8. Ditylenchus gallaeformans sp. n.
and D. drepanocercus clustered together with the highest
posterior probability. Phylogenetic analysis of rRNA gene
sequences revealed that these species are neither closely
Fig. 6. Gel with specic amplicons obtained from PCR with
species-specic primers. A: PCR with Ditylenchus gallaefor-
mans sp. n. specic primer (TW81 + D_gall primers); B:
PCR with D. drepanocercus species-specic primer (TW81 +
D_drep primers). Lanes: M: 100 bp DNA ladder (Promega); 1:
D. drepanocercus; 2-7: D. gallaeformans sp. n. (2-CA45; 3-
CA124; 4-CA131; 5-CA121; 6-CA129; 7-CA128); 8: control
without DNA.
related to the D. dipsaci species complex or to D.
destructor.
Thus, the BI analysis of the rRNA gene sequences
showed that Ditylenchus might be a paraphyletic taxon
including several evolutionary independent lineages. The
molecular groupings are congruent with those proposed
by Fortuner (1982), Sumenkova (1982) and Siddiqi
(2000), who divided the genus into several morpholo-
gical and biological species groups. The rst main group
constitutes plant-parasitic species of Ditylenchus with the
representatives of Anguininae, which have almost lost
their primitive trait of fungal feeding. The other group,
named as the D. triformis group by Siddiqi (2000), in-
cluded D. destructor Thorne, 1945 and unidentied (pos-
sibly fungal-feeding Ditylenchus). Identity of the sam-
ple identied in the tree as D. brevicauda (Micoletzky,
1925) Filipjev, 1936 might still be doubtful as Fortuner
(1982) and Brzeski (1991) considered this species as im-
perfectly described and regarded it as species inquirenda.
14 Nematology
Ditylenchus gallaeformans sp. n. from Brazil and Costa Rica
Fig. 7. The 50% majority rule consensus tree from Bayesian analysis generated from the partial 18S rRNA gene sequence dataset
using the GTR + I + G model and showing the relationships of Ditylenchus gallaeformans sp. n. and D. drepanocercus with other
Anguinidae. Posterior probabilities more than 70% are given for appropriate clades.
On the other hand, we cannot exclude the possibility that
the observed paraphyly could be the result of the use of
an inappropriate model of evolution for rRNA or possible
mistakes in identication. The phylogenetic hypothesis of
Ditylenchus as a monophyletic taxon should be carefully
tested in the future using several genetic markers and ad-
ditional species of Ditylenchus.
BIOLOGICAL CONTROL POTENTIAL
Only a few nematode species have been investigated so
far as potential biocontrol agents of weeds. All of these
nematodes belong to the Anguinidae and are parasites
of aerial plant parts, producing deformities and galls on
their hosts (Parker, 1991). Such nematodes belong to three
genera: Orrina Brzeski, 1981, Mesoanguina Chizhov &
Subbotin, 1985 and Ditylenchus. The nematode species
and their target weeds are: O. phyllobius (Thorne, 1934)
Brzeski, 1981 for Solanum elaeagnifolium Cav. (Robin-
son et al., 1978; Skinner et al., 1980; Parker, 1986); M.
picridis (Kirjanova, 1944) Chizhov & Subbotin, 1985 for
Acroptilon repens (L.) DC. (Watson, 1986); M. amsinck-
iae (Steiner & Scott, 1935) Chizhov & Subbotin, 1985 for
Amsinckia spp. (Pantone, 1987) and D. drepanocercus, for
M. calvescens (Seixas et al., 2004a, b).
Ditylenchus gallaeformans sp. n. is regarded as having
good potential for use as a classical biological control
agent against the weeds M. calvescens and C. hirta in
the Pacic islands. These two important weed species of
the Melastomataceae were found to be infected by this
nematode in natural habitats and infection sometimes led
to signicant damage on both weeds in the eld, also in
laboratory tests (Santin, 2008). The nematode is already
considered as being of particular interest for use as a
classical biological control agent against M. calvescens
and also C. hirta as these two very important weed species
Vol. 00(0), 2012 15
R.D.L. Oliveira et al.
Fig. 8. The 50% majority rule consensus tree from Bayesian analysis generated from the D2-D3 expansion segments of 28S rRNA gene
sequence dataset using the GTR+I +Gmodel and showing the relationships of Ditylenchus gallaeformans sp. n. and D. drepanocercus
with other Anguinidae. Posterior probabilities more than 70% are given for appropriate clades.
have been observed to be attacked by D. gallaeformans
and signicantly damaged both in the eld and in tests
under controlled conditions (Santin, 2008). Although the
host-range of the new nematode species is relatively wide
within the Melastomataceae (but restricted to this family)
(Dietrich, 2006; Santin, 2008), in the case of Hawaii, in
particular, this is of little concern as there are no native
Melastomataceae in Hawaii nor economically important
exotic cultivated melastomes. The biocontrol potential of
this new species has been extensively investigated by
Dietrich (2006) and Santin (2008) and results of this
research will be published separately.
Acknowledgements
This study includes part of a dissertation (Diplomarbeit
im Studiengang Biologie) presented by A. Dietrich to the
Institut fr Panzenkrankheiten, Leibniz Universitt Han-
noveran, and was performed with nancial support from
USGS BRD Pacic Islands Ecosystem Research Center,
National Park Service and the Research Corporation Uni-
versity of Hawaii, CNPq (Conselho Nacional do Desen-
volvimento Cientco e Tecnolgico) and CAPES (Coor-
denao de Aperfeioamento de Pessoal de Nvel Supe-
rior). The authors acknowledge A. McClay (McClay Eco-
science, Canada) for making the pioneering observation
of the nematode associated with the galls and for sharing
that information with the authors and hence stimulating
the present work.
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