© Benaki Phytopathological Institute
Hellenic Plant Protection Journal - Special Issue
16
University of Athens, Department of Crop Science, Laboratory of Plant Pathology,
75 Iera Odos Str., GR-118 55 Athens, Greece.
3
Department of Plant Pathology, USDA,
Agricultural Research Service, Tucson, AZ 85721, USA
One of the most efficient strategies to re-
duce the aflatoxin levels at pre-harvest lev-
el in several crops is the application of na-
tive biological control agents in the field.
One of the mechanisms of this technique is
based on the competitive exclusion of the
aflatoxigenic fungi
Aspergillus flavus
and
A.
parasiticus
by applying non-toxigenic micro-
organisms such as bacteria, yeasts or non-
toxigenic Aspergillus strains. The aim of the
present study was the evaluation of 136 non-
toxigenic strains of the genus
Aspergillus
, that
were isolated from pistachios of the typical
Greek variety “Aegina” collected from sever-
al orchards in Greece. The major goal is to re-
duce aflatoxin contamination from the field
by selecting and applying the most suitable
non-toxigenic strains. For that purpose, the
Greek isolates were DNA-characterized and
grouped with the method of Simple Se-
quence Repeats (SSRs) or Microsatellites us-
ing a multiple set of primers. Based on SSRs
results, the Greek strains were grouped in
20 different vegetative compatibility groups
(VCGs), whereas 65% of the total number of
isolates grouped to the same VCG. Next, afla-
toxinand cyclopionic acid (CPA) gene clusters
were investigated for possible indels using a
multiplex set of primers with the PCR meth-
od. The results showed indels in 7 Aspergil-
lus strains either in aflatoxin or in CPA gene
cluster. Also, the efficacy of representative
strains from different VCGs in aflatoxin re-
duction was evaluated with in vitro competi-
tion tests in autoclaved maize kernels. Com-
petition tests showed that treatments with 2
non-toxigenic strains from different VCG re-
sulted in 80% aflatoxin reduction compared
to control (toxigenic strain alone). The above
results demonstrate that some of the isolat-
ed Greek non-toxigenic Aspergillus strains
can be used as potential biocontrol agents
in pre-harvest stages in several aflatoxin sus-
ceptible crops in Greece.
Molecular characterization of the
cyp51
,
mdr
and
aflR
genes and the
effect of DMI resistance mechanisms on fitness parameters and aflatoxin
production in
Aspergillus parasiticus
E.G. D
OUKAS
1
, A.N. M
ARKOGLOU
1
, J.G. V
ONTAS
2
and B.N. Z
IOGAS
1
1
Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., GR-
118 55 Athens, Greece.
2
Faculty of Applied Biology and Biotechnology, Department
of Biology, University of Crete, GR-714 09 Heraklion, Greece
Aspergillus parasiticus
mutant strains resis-
tant to DMIs were isolated at a high muta-
tion frequency after UV-mutagenesis and
selection on media containing flusilazole.
Two different resistant phenotypes, char-
acterized as R
1
and R
2
on the basis of their
aflatoxigenic ability, were identified. All R
1
mutant strains produced aflatoxins at con-
centrations significantly higher (up to 3-fold)
than the wild-type parent strain on yeast ex-
tract sucrose medium, whereas the majori-
ty of the mutant strains (R
2
phenotype) had
lost their aflatoxigenic ability. Real-time PCR
analysis of the expression levels of the
aflR
gene, a pathway transcriptional regulato-
ry gene in aflatoxin biosynthesis, showed
that this gene was not expressed in R
2
mu-
tant strains tested. Study of fitness-deter-
mining parameters showed that most flusi-
lazole-resistant mutant strains had mycelial
growth rate, sporulation and spore germi-
nation rates lower that the sensitive strain.
Cross-resistance studies with other fungi-
cides showed that all R
1
mutant strains were
also resistant to the DMIs imazalil and tebu-
conazole, but retained their parental sensi-
tivity to fungicides affecting other metabolic
pathways and/or cellular processes. Contrary
