International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 4(4) pp. 97-102, April, 2013
Available online http://www.interesjournals.org/IRJPS
Copyright © 2013 International Research Journals
Full Length Research Paper
Molecular characterization and phylogenetic analysis of
turnip mosaic virus (TuMV) in Erysimum linifolium L. in
Italy
*1
Maria Grazia Bellardi, 1Lisa Cavicchi, 2Angelo De Stradis, 3Stefano Panno, 3Salvatore Davino
*1
Department of Agricultural Science, Alma Mater Studiorum, University of Bologna, Bologna, Italy
2
Istituto di Virologia Vegetale del CNR, Unità organizzativa di Bari, Bari, Italy
3
Department of Agricultural and Forestry Science, University of Palermo, Palermo, Italy
Abstract
In the Summer of 2012, Erysimum linifolium L. pot plants produced at an ornamental grower in Liguria
region (northern Italy), showed unusual virus-like disease of dark mottle and stripes on mauve-purple
petals. A virus was mechanically transmitted from symptomatic flowers to several test plant species
belonging to Chenopodiaceae and Brassicaceae families. This virus was identified as an isolated of
turnip mosaic virus (TuMV) by PAS-ELISA analysis, electron microscopy negatively stained crud
extracts and immuno-electron microscopy (IEM) tests. In the naturally infected E. linifolium plants,
TuMV occurred alone, since any other viruses either by electron microscopy or mechanical
inoculations were detected. By applying RT-PCR a fragment of 862 bp was amplified corresponding to
all coat protein (CP). The comparison of CP gene showed no correlations between their genetic
variation and geographical origins. The diversity in southern Europe appeared very low, most likely due
to the rapid growth of TuMV in relation to trade between different Countries. The consequent exchange
of infected propagation material shows that some lineages are adapted to particular crop species, and
that recombination is a significant generator of the genetic diversity in populations of this virus. This is
the first report of TuMV in E. linifolium worldwide.
Keywords: Aegean wallflower, Flower colour breaking, TuMV, Diagnosis, Molecular characterization.
INTRODUCTION
Erysimum linifolium L. (sin. Cheiranthus linifolium L.)
(Brassicaceae), or Aegean wallflower, native to the
Mediterranean region, is an evergreen perennial
ornamental shrub cultivated in Liguria region (northern
Italy) as pot flower plant producing masses of distinctive
pink through purple-mauve blossoms during the summer
months. This species growths to no more than 80 cm, so
it is used in limited spaces, such as rock gardens or in
mixed garden borders. Up to now Erysimum genus has
been indicated as susceptible to erysimum latent virus
(ELV), a tymovirus which was first isolated and described
*Corresponding Author E-mail: mariagrazia.bellardi@unibo.it
by Shukla and Schmelzer (1972) in symptomless E.
helveticum (Jacq.) D.C. growing in East Germany. In
addition, in 2012, a phytoplasma-like disease consisting
of leaf rosetting and growing reduction, was observed in
a few pot-plants of E. linifolium at an ornamental grower
of Albenga area (Liguria region). Direct PCR (polymerase
chain reaction), nested-PCR and RFLP (restriction
fragment length polymorphism) analyses allowed the
identification only in symptomatic plants of phytoplasmas
belonging to subgroup 16SrI-B (‘Candidatus phytoplasma
asteris’) (Paltrinieri et al., 2012).
In the Summer of 2012, some plot plants at blooming
stage of E. linifolium cultivated in the same area of
Albenga, were found to show virus-like symptoms
consisting of conspicuous dark mottle on all petals
(colour breaking); leaves were normal in colour and size
98 Int. Res. J. Plant Sci.
(figure 1). Considering that in the last decade the
economic importance of ornamental diseases associated
with viruses in Italy increased, especially in Liguria region
(Bellardi et al., 2011; Restuccia et al., 2011; Parrella et
al., 2012), specific studies were carried out to verify the
ethiology of flower colour breaking in this brassicacea
species. The present report describes the first natural
detection, identification, molecular characterization and
phylogenetic analysis of turnip mosaic virus (TuMV) in E.
linifolium.
MATERIALS AND METHODS
To detect virus infection in symptomatic petals of E.
linifolium, mechanical inoculations on herbaceous plants,
electron
microscope
observations
of
“leaf-dip”
preparations, serology (Protein-A Sandwich-ELISA: PASELISA; and Immuno-Electron Microscopy: IEM) were
employed. In particular, mechanical inoculations were
done by grinding symptomatic and asymptomatic
(control) petal tissue in 0.03 M cold phosphate buffer pH
7.0 containing 3% (w/v) polyethylene glycol (PEG, mol.
wt 6,000). Several herbaceous species, chosen among
those more susceptible to viruses infecting Brassicaceae
species, belonging to Chenopodiaceae, Lamiaceae and
Brassicaceae, were inoculated. Leaf tissue was
examined for virus-like presence under a Philips
Morgagni 268 transmission electron microscope (TEM),
at 80 kV, by applying “leaf-dip” technique. The crud sap
of naturally infected E. linifolium plants and some
inoculated herbaceous species showing symptoms
(Chenopodium amaranticolor, Ruta graveolens) was
negatively stained with 2% uranyl acetate (UA) aqueous
solution. PAS-ELISA (Edwards and Cooper, 1985) and
IEM (Milne and Lesemann, 1984) techniques were done
by using a polyclonal antiserum to TuMV available at
Laboratory of Virology (DiPSA, University of Bologna),
obtained from Istituto di Virologia Vegetale (CNR; Turin,
Piemonte region).
Total RNAs from young leaves were extracted. For
each sample, approximately 100 mg of leaf tissue was
ground in an Eppendorf tube in the presence of 500 μl
extraction buffer (200mM Tris pH 8.5; 1.5% SDS; 300mM
LiCl; 1% sodium deoxycholate; 1% Igepal CA-630; 10mM
o
EDTA), the mixture was incubate at 65 C for 10 min, 500
μl of potassium acetate pH 6.5 was added and incubated
on ice for 10 min. After a centrifugation of 10 min at
maximum speed, 650 μl of supernatant was transferred in
a new tube and an equal volume of cold isopropanol was
added and the mixture was incubated for 1 hour at –
0
80 C. After a centrifugation of 10 min at high speed, the
pellet was washed with 70% ethanol and resuspended in
50 μl of diethylpyrocarbonate-treated (DEPC)-treated
water.
RT-PCR was performed in one-step reaction in a 25 μl
final volume containing 2 μl of total RNAs (template), 20
mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 0.4 mM
dNTPs, 4U of RNaseOut, 20 U of SuperScript II reverse
transcriptase-RNaseH and 2U of Taq DNA polymerase
(Invitrogen, Carlsbad, CA, USA), 1 μM of primers forward
CP-TuMV+ (5’-GCAGGTGAGACGCTTGATGC-3’) and 1
μM
of
primer
reverse
CP-TuMV(5’TAACCCCTTAACGCCAAGTAAG-3’) corresponding to
genome position 8759 and 9601 of TuMV isolalte UK1
(Acc. No. AF169561) respectively. RT-PCR was carried
out in a Peltier Thermal Cycler PTC 100 (M.J. Research
INC., Waltham, MA, USA), under the following conditions
and parameters: 42°C for 30 min, 94°C for 2 min, 35
cycles of 30s at 94°C, 30s at 60°C, and 50s at 72°C with
a final elongation of 4 min at 72°C.
TuMV CP population structure was preliminarily
estimated by single stranded conformation polymorphism
(SSCP) analysis (Rubio et al., 1996). Sample showed
simple pattern, composed of two bands corresponding to
the two DNA strands (data not shown). Finally, the
consensus nucleotide sequences of the CP were
sequenced in both directions with an ABI PRISM 3100
DNA sequence analyzer (Applied Biosystems).
Multiple nucleotide sequence alignment was
performed with the algorithm CLUSTAL W (Larkin et al.,
2007). For this reason 50 sequences of CP protein of
Turnip mosaic virus were retrieved from GenBank.
Accession number was reported in the figure 2. The
substitution model that best fit these sequence data (with
the lowest Bayesian information criterion) was calculated.
Phylogenetic relationships were inferred by the
maximum-likelihood method (Nei and Kumar, 2000) with
1000 bootstrap replicates to estimate the statistical
significance of each node (Efron et al., 1996). All of these
analyses were performed with the program MEGA 5
(Tamura et al., 2011).
The nucleotide sequence diversity (mean nucleotide
distances) of CP-TuMV gene was estimated within and
between different countries or geographical regions,
considered as subpopulations. Four clusters were
constructed with the sequences retrieved from GenBank,
exactly China, Japan, northern Europe and southern
Europe. Australia, India, South Korea and Taiwan had a
number of sequences not sufficient to create groups
statically significant.
To assess the genetic differentiation and the gene
flow level between subpopulations the statistic Fst value
(Weir and Cockerham, 1984) were calculated. This test
was performed with DNAsp 5.0 program (Librado and
Rozas, 2009). To study the role of natural selection, the
rate of synonymous substitutions per synonymous site
(dS) and the rate of nonsynonymous substitutions per
nonsynonymous site (dN) were analyzed separately.
Generally, in a protein, only nonsynonymous changes are
subjected to selection, as they can alter the protein
function or structure. The difference between dN and
dS provides information on the sense and intensity of
Bellard et al. 99
Figure 1. Flowers of E. linifolium healthy (A) and TuMVinfected (B): dark mottle is visible on the petals.
Figure 2 . The Phylogenetic model that best fit with
the sequences retrieved from GenBank was
Tamura-Nei model, assuming variable substitution
rates among nucleotide sites α=0.19. Phylogenetic
analysis inferred with program MEGA 5 shows high
homology with isolates GBR98 from England (Acc.No.
EU861593) and isolates UK1 from Spain (Acc. No.
AF169561) with 99.8 and 99.4 respectively.
100 Int. Res. J. Plant Sci.
Figure 3. Leaf dip (left) and Immuno-electron microscopy (right) techniques from crude
extracts of C. amaranticolor (A, B) and R. graveolens (C, D) plants inoculated with
symptomatic flowers of E. linifolium. The IEM technique shows the filamentous potyvirus-like
particles decorated by antibodies to TuMV. Bar = 100 nm.
selection, for these reason dN>dS indicates positive or
adaptive selection, while dN<dS indicates negative or
purifying selection, and dN=dS indicates neutral
evolution. These values were estimated by the PamiloBianchi-Li method (Pamilo and Bianchi, 1993),
implemented in the program MEGA 5.
RESULTS
By using symptomatic petals of E. linifolium, chloronecrotic lesions on Chenopodium murale and local
chlorotic spots 5 days after inoculation enlarging into redrimmed lesions after 2-3 weeks on C. amaranticolor,
were observed; systemic symptoms appeared on
Brassica oleracea botrytis f cimosa (diffuse mottle) and
R. graveolens (leaf malformation, mosaic, dark green
vein banding). White flowers of symptomatic R.
graveolens were also infected. Filamentous potyvirus-like
particles 720-800 nm in length were observed by TEM in
negatively stained of leaf tissue extracts from naturally
infected
E.
linifolium
plants,
symptomatic
C.
amaranticolor and R. graveolens plants. These particles
were decorated by antibodies to TuMV, but not by
antibodies to other potyvirures such as clover yellow vein
virus, potato virus Y, tobacco etch virus, and watermelon
mosaic virus, four potyviruses that are known to occur in
wild species in Italy (Figure 3). The identity of the virus
associated with the flower disease of E. linifolium was
confirmed by PAS-ELISA and further by RT-PCR using a
pair of primers reported in Materials and Methods that
amplified a fragment of 862 bp corresponding to all coat
protein (CP) while no amplicons were obtained from
healthy plants.
The sequence obtained was depositated in GenBank
under the accession number CK736587. The
Phylogenetic model that best fit with the sequences
retrieved from GenBank was Tamura-Nei model,
assuming variable substitution rates among nucleotide
sites α=0.19. Phylogenetic analysis inferred with program
MEGA 5 showed high homology with isolates GBR98
from England (Acc. No. EU861593) and isolates UK1
from Spain (Acc. No. AF169561) with 99.8 and 99.4
respectively. From the data obtained, no correlation was
found between genetic relationship and geographic
location (Figure 2).
Nucleotide sequence diversity (mean nucleotide
distances) of CP gene of TuMV between and within
different countries or geographical regions was calculated
using program MEGA 5. and was reported in table 1.
To assess the genetic differentiation and the gene
flow level between subpopulations, the statistic Fst value
Bellardi et al. 101
Table 1. Nucleotide diversities within and between geographical populations. Number of base substitutions per site from
averaging over all sequence pairs between and within each group are shown. Standard error estimate(s) are shown in the last
column. Analyses were conducted using the Tajima-Nei model [1]. The analysis involved 38 nucleotide sequences. Codon
positions included were 1st+3rd. All positions containing gaps and missing data were eliminated. Evolutionary analyses were
conducted in MEGA5 [2]. *Indicate nucleotide diversities within each group.
Subpopulation
China
Japan
N.Europe
S.Europe
N. of TuMV isolates
18
9
6
5
China
0,085±0,008*
Japan
0,124±0,011
0,120±0,010*
N.Europe
0,129±0,012
0,108±0,008
0,085±0,010
S.Europe
0,112±0,013
0,082±0,007
0,059±0,006
0,003±0,002*
Table 2. First values between different subgroups.
China
Japan
N.Europe
S.Europe
China
Japan
N.Europe
0.206
0.305
0.584
0.045
0.262
0.258
were calculated. Two subpopulations with a similar
distribution of sequence variants would give an Fst value
that is statistically no different from zero, whereas an Fst
value of one would indicate total separation.
In the table 2 were reported Fst value between the
subgroups China, Japan, northern Europe and southern
Europe that are statically relevant. Table shows a high
gene flow between Japan and Northern Europe and
between China and Japan. Normal gene flow were
reported between the others subgroups. Last we
investigate the role of natural selection calculating the
rate of dS and dN.
The coat protein gene of TuMV had dN and dS
values of 0,0169 and 0,2698, respectively, indicating
negative selection due to functional and structural
constraints, which occurs in proteins having attained a
high level of adaptation. The dN/dS ratio was 0.062.
DISCUSSION
TuMV has an RNA genome and infects a wide range of
plant species, mostly, but not exclusively, from the family
Brassicaceae, as reported in 2003 in Iran, where Petunia
hybrida, Chrysanthemum sp. and Zinnia elegans showing
leaf and flowers symptoms resulted TuMV-infected
(Farzadfar et al., 2005). As regarding Brassicaeceae, it is
probably the most widespread and important virus
infecting both crop and ornamental species of this family,
and occurs in many parts of the world including the
temperate and tropical regions of Africa, Asia, Europe,
Oceania and North-South America (Provvidenti, 1996).
Concerning leaf symptomatology on vegetable and/or
ornamental brassicaceae species, TuMV causes mosaic,
mottling, black necrotic spots or ringspots on cabbage,
cauliflowers, Brussels sprouts, turnip mustard, Chinese
S.Europe
cabbage, etc., whereas on stock (Matthiola incana)
induces conspicuous “broken” flowers, with yellow stripes
and flecks in the petals of red coloured varieties (Bahar et
al., 1985). As regarding Erysimum (sin. Cheiranthus)
genus, in 2003 some wallflower plants (C. cheiri), grown
in commercial glasshouses in the Markazi province of
Iran, and showing mottle, leaf yellows and malformation,
were found naturally infected by TuMV (Farzadfar et al.,
2005). Up to now, any investigation has involved Aegean
wallflower (C. linifoliuum, sin. Erysimum linifolium) which
can be now included in the list of TuMV natural host.
TuMV belongs to the genus Potyvirus. This is the
largest genus of the largest family of plant viruses, the
Potyviridae (Ward et al., 1995), which itself belongs to the
picorna-like supergroup of viruses of animals and plants.
TuMV, like other potyviruses, is transmitted by aphids in
the non-persistent manner (Shukla et al., 1994). All
potyviruses have flexuous filamentous particles 700±750
nm long, each of which contains a single copy of the
genome, which is a single-stranded positive sense RNA
molecule about 10000 nt long. The genomes of
potyviruses have a single open reading frame that is
translated into a single large polyprotein, which is
hydrolysed, after translation, into several proteins by
virus-encoded proteinases (Riechmann et al., 1992). The
genomes of the Canadian (Ca) (Nicolas and Laliberte,
1992) and Japanese (1J) (Ohshima et al., 1996) isolates
of TuMV are 9830 and 9833 nt in length and have single
open reading frames which encode polyproteins of 3163
and 3164 amino acids, respectively. Of all potyviral
genes, that encoding the coat protein and situated at the
3«-end of the genome, has been most frequently studied
for its genetic diversity. In the study reported here, TuMV,
collected from naturally infected plants in Liguria, CP
gene sequenced and the comparisons of these
sequences show no correlations between their genetic
102 Int. Res. J. Plant Sci.
variation and geographical origins. It would seem that
gene flow is correlated with trade between the different
countries. Unlike other RNA viruses from the analyses
carried out can be seen as the intraspecific diversity
appears to be quite high for this virus. It is clear that the
diversity in southern Europe is very low (see table n. 2),
most likely due to the rapid growth of this virus in relation
to trade between different Countries and the consequent
exchange of infected propagation material show that
some lineages are adapted to particular crop species,
and that recombination is a significant generator of the
genetic diversity in populations of this virus.
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