Molecular identification of a 16SrIII-B phytoplasma

associated with cassava witches broom disease

Daniela Flres & Isolda Cristina Haas &
Maria Cristina Canale & Ivan Paulo Bedendo
Accepted: 27 June 2013
#KNPV 2013
Abstract Cassava plants displaying typical symptoms
commonly associated with phytoplasmas, such as
witches broom, general stunt, leaves with chlorosis,
deformation, and reduced size, were observed in fields
located in the state of So Paulo, Brazil. Molecular
analysis using specific primers evidenced that the phy-
toplasma found in all positive samples was affiliated with
group 16SrIII (X-disease group). Based on the virtual
RFLP patterns and similarity coefficient calculations, the
phytoplasma was classified as a member of the group
16SrIII, subgroup B (16SrIII-B). Phylogenetic analysis
showed this phytoplasma as closely related to the refer-
ence strain for the subgroup 16SrIII-B. Representatives
of 16SrIII-B have been described in several cultivated
species in Brazil and cassava is an additional host species
for this phytoplasma.
Keywords Cassava
.
Detection
.
Phytoplasma
.
Witches broom
Cassava (Manihot esculenta) serves as an important
source of carbohydrate for inhabitants of the tropical
regions, where it is used for human and animal nutrition
(FAO 2012). Cassava crops are distributed throughout
all Brazilian states and the country is the second among
the worlds largest producers, with an estimated annual
yield of 24.3 million tons (FAO 2012). Diseases caused
by various infectious agents may provoke serious losses
to the crops, thereby representing a limiting factor for
production and since it is vegetatively propagated path-
ogens may be disseminated with cuttings.
Phytoplasmas have been associated with an important
disease known as cassava witches broom in diverse
tropical regions, mainly in Central and South America,
represented by Cuba (Arocha et al. 2009b) and Brazil
(Lozano 1992), and in the African continent, represented
by Uganda (Arocha et al. 2009a). In Brazil, this disease
has been described in the 1940s in the southeast region
(Silberschmidt and Campos 1944). Later, it has been
observed in plants grown in the central (Kitajima and
Costa 1971), northeast (Mariano et al. 1991) and north
regions (Lozano 1992). In areas of the northeast region,
the disease may be present in 85 % of the fields and
provoke losses up to 70 % (Fukuda et al. 1990). In
this region, in savanna areas with elevation around
900 m.a.s.l., temperature averages 20 C, and semi-acid
soils (pH around 5.0), cassava witches broom may
cause up to a 90 % reduction in root yield (Lozano
1992). The disease also provokes yield losses of cuttings
for planting, considering that affected plants display
reduced size and excess budburst (EMBRAPA 2012).
Recently, the disease was observed in fields located
in the state of So Paulo (SP), southeast region, Brazil.
The symptoms were characterized by a generalized
stunting, shoot proliferation, foliar chlorosis and leaf
malformation (Fig. 1). The present study investigated
the association of symptomatic cassava plants with phy-
toplasma, which was molecularly and phylogenetically
analyzed.
Plants were sampled in fields located in the munic-
ipalities of Piracicaba, SP (25 symptomatic and 10
asymptomatic) and Bragana Paulista, SP (21 symptom-
atic and 10 asymptomatic). Total DNA was extracted
according to Doyle and Doyle (1987) and with DNeasy
Eur J Plant Pathol
DOI 10.1007/s10658-013-0250-3
D. Flres
:
I. C. Haas
:
M. C. Canale
:
I. P. Bedendo (*)
Departamento de Fitopatologia, ESALQ-USP,
C. P. 09, 13418-900 Piracicaba, SP, Brazil
e-mail: ibedendo@usp.br
Plant Mini Kit (Qiagen). Phytoplasma detection was
performed by nested PCR assay using the primer pairs
P1/Tint and R16F2n/16R2, as previously described
(Rapussi et al. 2012). DNA from asymptomatic plants
served as a negative control, while the positive control
was represented by DNA of 16SrIII-J phytoplasma
(Montano et.al. 2000).
Molecular identification was carried out by nested
PCR using P1/Tint primers followed by R16(III)F2/R1,
specific for phytoplasmas of the 16SrIII group (Lee
et al. 1994), and R16(I)F1/R1, which may amplify
phytoplasmas of the groups 16SrI, 16SrII, 16SrXII
and 16SrXV (Lee et al. 1994; Cieslinska and
Kruczinska 2011). The controls were represented by
DNA extracted from maize and chayote plants infected
with phytoplasmas belonging to the groups 16SrI-B
(Lee et al. 1998) and 16SrIII-J (Montano et al. 2000),
repectively.
The DNA products of 1.2 kb generated by nested
PCR with the P1/Tint and R16F2n/R16R2 primers were
subjected to RFLP analysis with restriction enzymes
(AluI, BstUI and HhaI) (Invitrogen) and electrophoresed
with a 5 % polyacrylamide gel, stained with Sybr Safe
(Invitrogen). The RFLP profiles obtained for the phyto-
plasma detected in the cassava were compared with the
profiles described (Lee et al. 1998).
The gene 16S rRNA of the isolates Md1 (Piracicaba)
and BG13 (Bragana Paulista) were cloned using the
pGEM-T Easy Vector System I (Promega), transformed
into the Escherichia coli DH5 strain, and sequenced
using the primers pair SP6/T7 (Malembic-Maher et al.
2008). The DNAfragments of cassava phytoplasma were
Fig. 1 Symptomatic cassa-
va plants: a general stunt
(plants with 5 months after
planting of the cuttings), b
witches broom, and leaves
with chlorosis, deformation,
and reduced size
Fig. 2 RFLP analysis of the 16S rRNA gene of the isolates Md1
(Piracicaba-SP) and BG13 (Bragana Paulista-SP) after nested
PCR amplification with P1/Tint and R16F2n/R16R2 and
digestion with enzymes AluI, BstUI, and HhaI. Lanes: M (marker =
174RF HaeIII. Biolabs); Ch = positive control (chayote witches
broom phytoplasma)
Eur J Plant Pathol
aligned, compared among themselves, with the se-
quences of phytoplasmas of distinct groups and with
the 16SrIII subgroups representatives using DNA analy-
sis programs (Phred phrap, Bioedit, MEGAand Multiple
Sequence AlignmentCLUSTALW).
A phylogenetic tree was constructed with the se-
quences from both isolates from cassava plants (Md1
and BG13) and the sequences of the 16S rRNA gene
belonging to different phytoplasmas (Fig. 3) using the
ClustalW (Multiple Sequence Alignment, Cambridge,
UK) program with the Neighbour-Joining method.
A similarity coefficient (F) was calculated for each
pair of phytoplasma based on restriction patterns pro-
duced by computer-simulated RFLP analysis of the 16S
Fig. 3 Phylogenetic tree constructed on the basis in 16S rRNA
gene sequences from phytoplasmas found in association with
cassava witches broom (Md1 = CaWB-Br01 and BG13 =
CaWB-Br02) and representatives of diverse groups and
subgroups. Acholeplasma laidlawii was used as outgroup. The
numbers on the branches represent bootstrap values for 1,000
replicates
Eur J Plant Pathol
rRNA gene, according to Wei et al. (2007). In this study
were used sequences of approximately 1.25 Kb belong-
ing to both isolates from cassava phytoplasma and se-
quences representing various phytoplasmas of the 16SrIII
group found in the GenBank database (Fig. 3).
Based on infected plants that exhibited typical symp-
toms of the disease, the incidence levels were estimated
to be about 15 % in Piracicaba and approximately 80 %
in Bragana Paulista. Phytoplasma was detected in 76 %
of symptomatic plants sampled in Piracicaba and 95 %
of those collected in Bragana Paulista, through ampli-
fication of DNA fragments of 1.2 Kb generated by the
nested PCR assays. No amplification was obtained for
DNA extracted from asymptomatic plants.
Nested PCRassays primed by group-specific primers
generated a product of 0.8 Kb with DNA from all
symptomatic plants, thereby indicating the presence of
a 16SrIII phytoplasma group. In contrast, no amplifica-
tion occurs from the PCR primed by the group-specific
primers R16(I)F1/R1.
Three phytoplasma isolates found in the cassava
samples collected in each region were selected for
RFLP analysis, after their identification by PCR using
a group-specific primer pair. The collective RFLP
patterns generated from the digestion of the 1.2 Kb
DNA fragments with three restriction enzymes were
indistinguishable for all isolates present in the six sam-
ples. One isolate from each region (Piracicaba-SP and
Bragana Paulista-SP) was selected as a representative
of all six isolates sampled. The restriction patterns
displayed by these isolates are shown in Fig. 2. The
patterns from the cassava isolates were identical to
those produced by the chayote witches broom phyto-
plasma, excluding HhaI since this enzyme is a key
enzyme for the identification of the reference phyto-
plasma for the 16SrIII-J subgroup.
The 16S rRNA gene of the cassava phytoplasma
represented by the isolates Md1 (Piracicaba) and BG13
(Bragana Paulista) was used for sequencing. Three
clones of each isolate were sequenced and a majority
consensus sequence was selected for each isolate, as no
polymorphism was observed. Therefore, the consensus
sequences of approximately 1.25 Kb corresponding to the
isolates Md1 and BG13 were designated by CaWB-Br01
(cassava witches broom- Brazil 01) and CaWB-Br02
(cassava witches broom- Brazil 02), which were depos-
ited in GenBank under the accession numbers GU193976
and GU193977, respectively. The DNA sequences from
Table 1 Values of similarity coefficients (F) calculated from restriction patterns generated by virtual Restriction Fragment Length
Polymorphism (RFLP)
Subgroup A B E F G H I J P1 Q R1 PPT-M ChD-T CaWB CaWB
Br01 Br02
CX-A 1
CYE-B 0.93 1
SP1-E 0.96 0.94 1
MW1-F 0.88 0.93 0.89 1
WWB-G 0.96 0.91 0.95 0.87 1
PoiBI-H 0.96 0.94 0.97 0.89 0.97 1
VGY-I 0.86 0.94 0.97 0.89 0.95 0.97 1
ChWB-J 0.89 0.96 0.9 0.96 0.88 0.9 0.9 1
Dan Vir-P1 0.86 0.93 0.87 0.93 0.84 0.87 0.87 0.94 1
BRWB7-Q 0.85 0.95 0.89 0.95 0.87 0.89 0.89 0.96 0.93 1
CirWL-R1 0.82 0.89 0.83 0.87 0.81 0.83 0.83 0.87 0.85 0.87 1
PPT-M 0.81 0.86 0.85 0.94 0.85 0.85 0.85 0.94 0.88 0.88 0.83 1
ChD-T 0.78 0.85 0.79 0.87 0.76 0.76 0.79 0.86 0.89 0.85 0.77 0.81 1
CaWB-Br01 0.93 1 0.94 0.93 0.91 0.94 0.94 0.96 0.93 0.95 0.89 0.86 0.85 1
CaWB-Br02 0.93 1 0.94 0.95 0.91 0.94 0.94 0.96 0.93 0.95 0.89 0.86 0.85 1 1
CX Canadian X-disease, CYE clover yellow edge, SP1 spirea stunt, MW1 milkeed yellows, WWB walnut witches broom, PoiBI
poinsettia branch-inducing, VGY Virginia grapevine yellows, ChWB chayote witches broom, BRWB7 black raspberry witches broom,
DanVir dandelion virescence, CirWL cirsium white leaf, PPT-M potato purple top, ChD-Tcherry decline, CaWB cassava witches broom
Eur J Plant Pathol
Md1 and BG13 demonstrated 99.9 % similarity in
relation to the sequence of Clover Yellow Edge phy-
toplasma (CYE), the reference strain for the subgroup
16SrIII-B (GenBank AF175304). In agreement, the
similarity coefficient (F) calculated between these iso-
lates and the CYE phytoplasma, based on the virtual
patterns, was equal to 1.0 (Table 1). Moreover, the
phylogenetic tree showed that the isolates Md1 and
BG13 are closely related to the representative of sub-
group 16SrIII-B (Fig. 3).
The initial assumption that the symptoms of stunting,
shoot proliferation, foliar chlorosis and leaf malforma-
tion were associated with phytoplasma was demonstrat-
ed by the PCR assays. These symptoms were coincident
with the first description of the disease observed in the
state of So Paulo (Silberschmidt and Campos 1944).
Additionally, the molecular evidences of the association
between phytoplasma and cassava witches broom
presented here are in agreement with previous descrip-
tions of this disease in other regions, in which
phytoplasmas were found in symptomatic plants by
using electron microscopy (Kitajima and Costa 1971;
Mariano et al. 1991).
More recently, distinct phytoplasmas have been re-
ported in association with cassava diseases. In Pacific
Island (Davis et al. 2005) and Cuba (Arocha et al.
2009b), a phytoplasma affiliated with group 16SrI
(subgroup not identified) and 16SrI-C, respectively,
were found in cassava plants that exhibited stunting
and foliar yellowing. However, in Uganda, a phyto-
plasma belonging to group 16SrII was characterized in
association with cassava plants displaying similar
symptoms (Arocha et al. 2009a). The present investi-
gation reveals that a different phytoplasma (16SrIII-B)
is present in cassava exhibiting symptoms of witches
broom. The identification was shown according to the
schemes established for the molecular characterization
and classification of phytoplasmas in the diverse
groups and subgroups (Lee et al. 1994; Lee et al.
1998; Wei et al. 2007). Preliminary evidence of the
presence of a phytoplasma affiliated with group 16SrIII
in cassava has been reported in plants sampled in the
northeast region (Barros et al. 1998).
Cassava frogskin disease (CFSD) is also associated
with a group 16SrIII phytoplasma, but belonging to
16SrIII-L subgroup (Alvarez et al. 2009). The symptoms
present in affected plants are distinct of those observed
for witches broom associated with 16SrIII-B phytoplas-
ma. Characteristically, the symptoms of CFSD are
observed in the roots, which show a woody aspect, deep
lesions and a thickened peel that is cork-like (Alvarez
et al. 2009). According to these authors, although some
cultivars may also exhibit leaf symptoms such as mosaic,
chlorosis, and curvature in leaf margins, generally the
aerial parts of diseased plants are more vigorous and
better developed than those of healthy plants. In contrast,
the roots of plants sampled in the present study did not
show such types of symptoms and the aerial parts
exhibited stunting, with presence of leaves and branches
of reduced size. Thus, there is strong evidence that these
diseases are caused by distinct phytoplasmas.
Phytoplasmas classified in the group 16SrIII, mainly
those belonging to subgroups 16SrIII-B and 16SrIII-J,
are most frequently identified in association with a
diversity of diseases that affect different species grown
in various Brazilian geographic areas, including cauli-
flower, chayote, eggplant, pumpkin, tomato, begonia,
Celosia spp. and China tree (Rapussi et al. 2012). Based
on our findings, in addition to these cultivated species,
cassava represents an additional host species of a phy-
toplasma affiliated with the 16SrIII-B subgroup.
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