© Benaki Phytopathological Institute
Abstracts - 16th Hellenic Phytopathological Congress
19
Inactivation of the
VdVeA
(Velvet A) gene in
Verticillium dahliae
and investigation of its role in the physiology and pathogenicity of the
fungus
A. A
NTONIADI
and D.I. T
SITSIGIANNIS
Agricultural University of Athens, Department of Crop Science, Laboratory of Plant
Pathology, 75 Iera Odos Str., GR-118 55 Athens, Greece
Velvet complex, in association with a third
protein, VelB. The aim of this study was to
elucidate the role of the orthologous gene
of
A. nidulans, VdVeA,
in the virulence and
morphology of the fungus
V. dahliae
. A gene
replacement strategy was applied by incor-
porating two genomic sequences of about
1000 bp before the start and stop codons re-
spectively, in the binary vector pGKO2. Be-
tween these two regions the geneticin cas-
sette was subcloned in order to replace the
VdVeA
gene after transformation. The inac-
tivation construct of
VdVeA
was introduced
into various strains of
V. dahliae
using the Ti
plasmid of
Agrobacterium tumefaciens
via a
double recombination event. The effect of
inactivation of the
VdVeA
gene on the phys-
iology and pathogenicity of the fungus was
explored in
in vitro
experiments and
in plan-
ta
virulence assays in several hosts.
Fast and accurate identification of
Fusarium oxysporum
formae
speciales
complex using High Resolution Melting (HRM) analysis
I. G
ANOPOULOS
1,2
, P. M
ADESIS
2
and A. T
SAFTARIS
1,2
1
Department of Genetics and Plant Breeding, School of Agriculture, Aristotle
University of Thessaloniki, GR-541 24 Thessaloniki, Greece.
2
Institute of Applied
Biosciences, CERTH, GR-570 01 Thermi, Thessaloniki, Greece
Verticillium dahliae
is a soilborne plant
pathogenic fungus causing the syndrome of
wilt diseases and posing a significant threat
to several annual and perennial crops world-
wide. The inability of common management
methods to control
V. dahliae
has led to the
investigation of molecular mechanisms that
might regulate its virulence. Studies have
previously shown that the fungus
V. dahliae
produces phytotoxins and other secondary
metabolites that induce cell death or oth-
er forms of host defence. It has been found
that in several species of
Fusarium
spp. and
Aspergillus
spp. the gene
veA
encodes a pro-
tein that can regulate the fungal secondary
metabolism, induce the differentiation of
fungal development in relation to light, and
regulate reproduction and pathogenicity.
Along with the VeA, a second protein called
LaeA forms a nuclear complex called the
The fungus
Fusarium oxysporum
consists
by a number of different strains which are
grouped together in to groups called for-
mae speciales making F. oxysporum a highly
complex species. This complexity is respon-
sible for the timely and difficult discrimi-
nate of the different Fusarium formae spe-
cials which is performed via biochemical
or phenotypic methods. Thus it is of para-
mount importance to develop novel, rapid,
and simple to perform identification meth-
ods. Herein, we describe the development
of a novel real-time PCR based assay [using
universal internal transcribed spacer (ITS)
primers] coupled with high-resolution melt-
ing (HRM) analysis for the identification and
discrimination of
F. oxysporum
formae
spe-
ciales
complex. The melting curve analysis
of the ITS amplicons succeeded in specifi-
cally classify all isolates into seven
F. oxyspo-
rum
formae
speciales
and generated sev-
en distinct HRM curve profiles. The smallest
DNA sequence difference recognized in this
study was one nucleotide. We conclude that
based on the mentioned results HRM curve
analysis of
Fusarium
ITS sequences is a sim-
