© Benaki Phytopathological Institute
Hellenic Plant Protection Journal - Special Issue
10
The quantification of plant pathogens using
DNA-based molecular tools can be mislead-
ing due to an inability to distinguish signals
originating from live and dead cells. Howev-
er, methods that use DNA-intercalating dyes
like propidium monoazide (PMA) have been
used to selectively remove cells with com-
promised cell membranes from the analysis,
which can be considered to be dead. These
dyes are nearly completely cell membrane-
impermeable and therefore can be selec-
tively used to modify only exposed DNA
from dead cells while leaving DNA from vi-
able cells intact. Once these dyes enter a
cell, they bind to DNA and can be covalently
crosslinked to it by light exposure. PCR am-
plification of such modified DNA is strong-
ly inhibited. In this study we evaluated the
suitability of PMA treatment to distinguish
between viable and nonviable plant patho-
gens. A PMA-qPCR combined assay was ap-
plied to viable and inactivated fungal patho-
gens. Cell suspensions were incubated with
PMA, and then exposed to light to secure the
intercalation of PMA with the DNA of dead
cells, and to inactivate any unbound PMA.
Treated cells were extracted and the rela-
tive ratios of live and dead cells were evalu-
ated by qPCR. After heat treatment and DNA
modification with PMA, all fungal species
tested showed an approximate 100- to 1000-
fold difference in cell viability as estimated
by qPCR analysis, which was consistent with
estimates of viability based on culturing.
Population genetic structure of
Phytophthora infestans
in Cyprus
L. K
ANETIS
, L. P
ITTAS
, D. T
SALTAS
and N. I
OANNOU
Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus
University of Technology, 3603 Limassol, Cyprus
A total of 521 isolates of
P. infestans
, the caus-
al agent of potato late blight, were collected
in Cyprus from 2009 to 2011. The scope of the
present study was the genotypic character-
ization of the local population of the micro-
organism including mating type and DNA
fingerprinting patterns using 12 microsatel-
lite markers. During the first two years of the
study, when a nationwide collection took
place, the presence of both mating types
was documented at a ratio of approximate-
ly 1:1. In 28.5% of the sampled fields both
mating types coexisted, suggesting the po-
tential for sexual reproduction. In addition,
13 genotypes of
P. infestans
were identified,
with prevailing types 13_A2, 2_A1 and 23_
A1. More specifically, genotype 13_A2, which
has been reported in many European coun-
tries and which is characterized by high ag-
gressiveness and resistance to the fungicide
metalaxyl-M, appeared frequently through-
out the sampling period. The 2011 sampling
was concentrated in a single potato field and
only genotypes of 13_A2 were identified.
Overall, the relatively low genetic variability
of the Cyprus populations of
P. infestans
indi-
cates the absence of sexual reproduction of
the microorganism, despite the existence of
both mating types. Monitoring the genetic
background of the local population may pro-
vide information on the appearance of sexu-
al reproduction of the pathogen, and the po-
tential invasion of new genotypes, primarily
from seed exporting countries.
Classification of Cretan
Verticillium dahliae
isolates to races and their
virulence characterization in differential hosts
A.E. P
OMPODAKI
1
, E.A. M
ARKAKIS
1
and E.K. L
IGOXIGAKIS
2
1
Laboratory of Plant Pathology, School of Agricultural Technology, Technological
Educational Institute of Crete, Stavromenos, GR-710 04 Heraklion, Crete, Greece.
2
Laboratory of Plant Pathology, Plant Protection Institute of Heraklion, Hellenic Agri-
cultural Organization “Demeter”, Mesa Katsampas, GR-710 03 Heraklion, Crete, Greece
