© Benaki Phytopathological Institute
Hellenic Plant Protection Journal - Special Issue
50
from eggplant was sequenced but epidemi-
ological data concerning the presence and
dispersal of the virus is still limited. The pur-
pose of the present study was the develop-
ment of a rapid and sensitive method of re-
verse transcription PCR (Real Time qRT-PCR)
to detect and quantify EMDV in plant tis-
sues and insect vectors. A 210 nucleotides
long conserved region of the polymerase
(L) gene was used as target of the assay.
In
vitro
synthesized EMDV-RNA transcripts of
known concentrationwere used for the eval-
uation of the system. Moreover, the extrac-
tion method of the viral RNA as well as the
conditions of qRT-PCR were optimized. The
amplification efficiency was 96.9% and the
linear range of quantification was from 20 to
2×10
8
RNA transcripts. The above assay was
applied to total RNA extracts from tissues of
eggplant, honeysuckle (
Lonicera japonica)
,
tomato, tobacco, caper (
Caparis spinosa)
, cu-
cumber,
Pittosporum tobira
and hibiscus (
Hi-
biscus syriacus
) in which the virus was suc-
cessfully detected. The developed method
proved to be a simple and reliable tool for
the detection and quantification of the virus
in host plants as well as in insect vectors.
Development of a semi-nested RT-PCR for the detection of ApLV and
study of its presence in Greece
I. F
OTIOU
, P.G. P
APPI
, V.I. M
ALIOGKA
and N.I. K
ATIS
Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural
Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24
Thessaloniki, Greece
Apricot latent virus
(ApLV) is a member of
the genus
Foveavirus
, family
Betaflexiviridae
,
and infects a wide range of plant species of
the family Prunoideae. It is spread in differ-
ent countries. Although it is usually latent,
in some host plants it causes symptoms but
without severe yield losses. The available
detection methods of ApLV are not reliable
as they do not detect all virus isolates. In this
study, we developed an improved detection
method of ApLV which was also used for
testing different plant species of the fam-
ily Prunoideae for the virus presence. For
this purpose, a semi-nested RT-PCR was de-
veloped using degenerate primers which
bind to conserved sequences of the 5’ un-
translated region of the viral genome and
the 5’-end of the RNA dependent RNA poly-
merase (RdRp) gene. The method was suc-
cessfully evaluated by using four charac-
terized virus isolates (Casserta 12, LA2, SB,
A18) and proved to have a wider detection
range compared to the available methods
as it detects all virus isolates tested during
this study. Finally, the developed method
was used to test 546 stone fruit samples (al-
mond, apricot, cherry, peach, ornamental
plum) from different regions of Greece for
the presence of ApLV. The virus was not de-
tected in any of them.
