© Benaki Phytopathological Institute
Hellenic Plant Protection Journal - Special Issue
38
atization, multiplexing etc.) and in the pro-
cessing of the vast amount of sequence data
generated, were many questions remain. In
the conceptual field, these developments
are taking us to a new, more integrated vi-
sion of viral ecology.
O
RAL
& P
OSTER
P
RESENTATIONS
Analysis of siRNAs using a new generation sequencing platform for the
detection and characterization of viruses and viroids present in a citrus
sample
A. O
LMOS
1
, C. V
ARVERI
2
, M.C. M
ARTÍNEZ
1
, E. B
ERTOLINI
1
, T. C
ANDRESSE
3,4
and M. C
AMBRA
1
1
Instituto Valenciano de Investigaciones Agrarias (IVIA). Plant Protection and
Biotechnology Center, Moncada-Naquera km5, 46113 Moncada, Valencia, Spain.
2
Benaki Phytopathological Institute, Laboratory of Virology, 8 St. Delta Str., GR-145 61
Kifissia, Greece.
3
INRA, UMR1332 BFP, BP81, 33883 Villenave d’Ornon cedex, France.
4
Université de Bordeaux, UMR 1332 BFP, BP81, 33883 Villenave d’Ornon cedex, France
The population of virus derived small inter-
fering (si) RNAs (21-24 nucleotides) induced
by Dicer processing of dsRNAs formed dur-
ing RNA virus replication can be sequenced
by Ion Torrent next generation sequencing
technology. This strategy has been success-
fully used to analyze a sample derived from
a lemon tree of the “Lemonodasos” area in
Poros (Trizinia, Prefecture of Piraeus) where
Citrus tristeza virus
(CTV) had been previously
detected. Initially, lemon samplesweregraft-
ed on seedlings of sweet orange cv. Madam
Vinous and then siRNAs were isolated. The
Ion Torrent platform allowed the determina-
tion of 432.632 sequences. The
de novo
bio-
informatic analysis confirmed the presence
of CTV by detecting several large contigs
of this virus species. The isolate present in
the sample was reconstructed by mapping
the specific siRNAs against several reference
isolates, which allowed the determination
of almost the full-length 19.251 nt genome
of this L192GR-CTV isolate. Only a small gap
of 18 nt was identified and was later deter-
mined by RT-PCR and direct sequencing.
Phylogenetic analysis of the L192GR isolate
revealed high molecular homology with
the Israeli VT-CTV isolate (GenBank Acc. No
EU937519.1) with 98% sequence identity. In
addition, the
de novo
analysis of siRNA se-
quences allowed the reconstruction of the
complete full-length genomes of
Citrus exo-
cortis viroid
and
Hop stunt viroid
and the re-
trieval of partial sequences of
Citrus viroid
III
and
Citrus viroid IV
. These results demon-
strate the great potential of this next-gen-
eration sequencing platform, opening new
possibilities for the diagnosis and character-
ization of citrus viruses and viroids.
Development of a nested RT-PCR for the detection of
Little cherry virus-1
and study of its presence in several host species
Α.T. K
ATSIANI
, Ε.V. D
ROUGKAS
, V.Ι Μ
ALIOGKA
and Ν.Ι.Κ
ATIS
Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural
Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24
Thessaloniki, Greece
Little cherry virus-1
(LChV-1) and
Little cherry
virus-2
(LChV-2), both members of the fam-
ily
Closteroviridae,
are associated with lit-
tle cherry disease. In previous surveys, only
