span class="fs6 ws2 v4"> of active ingredient.
In vitro produced grape leaves of cv. “Baresana” were
used to assess the sensitivity of E. necator isolates to
fungicides [2]. Leaves were standardized in age and size
to minimize any possible influence of such factors on the
growth of E. necator colonies.
Application of fungicides was carried out by dipping
leaves in fungicide suspensions at appropriate concentra-
tion containing 0.05% tween 20 for 1 min under gentle
shaking. Control experiments were conducted by im-
mersing leaves in sterile distilled water containing 0.05%
Tween 20. Leaves were then placed in 55-mm-diam Petri
dishes containing 10 ml of B0/2 substrate as described by
Miazzi et al. (1997) [15]. Petri dishes were left closed in
a laminar flow cabinet for 8 h before inoculation. Each
leaf was then inoculated at single point with about 15 -
20 conidia under a stereomicroscope at 50× magnifica-
tion. Inoculated leaves were then kept in a growth cham-
ber at 21˚C ± 1˚C and exposed, 16 hours per day, to the
light produced by a combination of 3 Osram L36W Cool
White lamps and 3 Silvanya Grolux F36W lamps.
After two weeks, diameter of fungal colonies was
measured with the aid of stereomicroscope. The effective
concentration at 50% (EC50) was calculated for individ-
ual isolates. Resistance factor (RF) was calculated ac-
cording to the formula RF = EC50 for the resistant isolate/
EC50 of sensitive isolates.
2.3. Molecular Biology Assay
The primers RSCBF1 and RSCBR2, designed by Ishii et
al. (2001) [16] on the ground of highly conserved regions
of cytb gene in fungi and proved specific for the cytb
gene in Sphaeroteca fusca, were used.
DNA was extracted from mycelium and conidia with
InstaGene Matrix (Bio-Rad Laboratories, USA). PCR
was performed in 25 l reaction mixtures containing 50
ng of total DNA, 2 mM MgCl2, 200 M each dNTP
(Promega, Madison, WI, USA); 10 mM Tris-HCl, pH 8.3;
50 mM KCl; 0.1% gelatin, and 1 U of Red Taq DNA
polymerase (Sigma, St Louis, Missouri, USA). PCR
reactions were performed in a thermal cycler (Gene Amp
PCR System 9700; Perkin-Elmer, Norwalk, USA) pro-
grammed as follow: 4 min at 94˚C, followed by 40 cycles
of 30 sec at 94˚C, 1 min at 52˚C, 1.5 min at 72˚C, and a
final extension stage of 7 min at 72˚C [16]. PCR products
were separated on 2% agarose gel (Bio-Rad Laboratories,
USA) in 0.5× TBE buffer (45 mM Tris-borate, 1 mM
Na-EDTA; pH 8) at 110 V for 120 min (Sub-Cell TM,
Bio-Rad Laboratories, USA), The gal was stained with 1
μg·ml1 ethidium bromide for 10 min and the bands were
recovered by mechanical excision of small gel plugs.
DNA fragments were eluted, purified using the Qiaex II
Gel Extraction Kit (QIAGEN, Germany) and sequenced
by MWG Biotech (Italy).
2.4. Generation of Azoxystrobin Resistant
Putative Mutants
Mono-conidial cultures of the G82 isolate, were grown in
vitro grapevine leaves treated with azoxystrobin. At 1-
month intervals, colonies were sub-cultured on leaves
treated with increasing concentrations (0.1, 1, 5, 10 and
20 μg·ml1) of the fungicide. After that cultures were
maintained on leaves treated with 10 or 20 μg·ml1
azoxystrobin.
3. Results
Twenty E. necator isolates representative of the tow ge-
netic groups (Table 1), were used to establish baseline
sensitivity to azoxystrobin. EC50 values ranged from less
than 0.1 to 0.3 μg·ml1 azoxystrobin. Colony growth was
inhibited at 0.1 to 6 μg·ml1 of the fungicide. Differences
in EC50 values among tested isolates were very low. MIC
values showed by isolates belonging to group B were
between 1 - 6 μg·ml1 azoxystrobin, were slightly higher
than that of group A isolates. MIC values of group A
isolates wer between 0.1 - 1 μg·ml1 azoxystrobin. It is
obvious that in most cases, the highest MIC value for
group A isolates corresponds to the lowest MIC value for
group B isolates.
DNA amplification with he primers RSCBF1 and t
Copyright © 2012 SciRes. AJPS
Observation on Baseline Sensitivity of Erysiphe necator Genetic Groups to Azoxystrobin
Copyright © 2012 SciRes. AJPS
1642
Table 1. Sensitivity of E. necator isolates belonging to the two genetic group to azoxystrobin.
Colonies diameter (mm) of 14-days-old colonies grown on leaves treated with
various concentrations (μg·ml1) of azoxystrobin
Genetic group Isolate
0 0.1 0.3 1 3 6
EC50 MIC
X1 14 10 3 0 0 0 <0.3 1
X156 7.6 1 2 0 0 0 <0.1 1
X165 3.3 2 0 0 0 0 <0.3 0.3
X167 0 0 0 0 0 <0.1 0.1
X215 5.6 3 1 0 0 0 <0.3 1
X217 4.3 3 0 0 0 0 <0.3 0.3
X221 4.5 4.3 1.5 0 0 0 <0.3 1
X236 4.6 0 0 0 0 0 <0.1 0.1
X280 4.5 3 3 0 0 0 <1 1
X283 6.3 5.3 2.5 0 0 0 <0.3 1
A
“Flag-shoot”
X292 2.6 1.5 0 0 0 0 <0.3 0.3
G82 11 10 5 2 0 0 <0.3 3
X161 5 2 2 0 0 0 <0.1 1
X185 7.6 3 2.6 2 0 0 <0.1 3
X186 7.6 4.3 2 1 0 0 <0.3 3
X233 5.3 3 1.5 0 0 0 <0.3 1
X234 5 3.3 2 0 0 0 <0.3 1
X255 6.3 3 2.6 2.6 2.3 1 <0.1 >6
X294-A 6.3 1.5 4.3 0 0 0 <1 1
B
“Ascospore”
X296 5.6 4.5 2.3 1 1 0 0.3 6
RSCBR2, of mono-conidial isolate X34, yielded 2 bands
of 286 bp (StI) and 224 bp (StII) (Figure 1).
The StI and StII amplicons were sequenced and ana-
lyzed with FASTA sequences in Organelles Library of
EBI GenBank. Our results showed that StI, but not StII,
had a high similarity with the sequences of the mito-
chondrial cytb of other fungi (e.g. Erysiphe graminis,
Magnaporthe grisea, Venturia inaequalis, Podosphaera
fusca, Saccharomyces cerevisiae). The similarity within
the best 100 scores was 68% - 87.7% identity and 69% -
87.7% for the un-gapped alignment (Figure 2).
The StI nucleic acid sequence was translated into
amino acid sequence using the ExPASy (Expert Protein
Analysis System, Swiss Institute of Bioinformatics,
Switzerland; available at website http://us.expasy.org/).
The resulting amino acid sequence was aligned with
those of other fungi. StI proved to contain amino acid
sequence from 53 to 162 of the cytb gene, no mutations
at G143A and F129L were present (Figure 2).
The G82 isolates, were used in experiments for the
production of azoxystrobin resistant mutants. Periodical
transfer of conidia onto grapevine leaves treated with
various concentrations of azoxystrobin resulted in 12
putative mutants of G82 (Table 2). The most 6 promis-
ing putative resistant mutants (G82-a, G82-d, G82-h,
G82-i, G82-j and G82-k) were maintained on leaves
treated with azoxystrobin at various concentrations (0,
0.1, 0.3, 1, 5, 10, 20, 100 μg·ml1). With the exception of
G82-i, EC50 values for the putative mutants were not
distinguishable from that of the parental G82 wild type
isolates (<0.1 μg·ml1). Although MIC values for 5 of
them (G82-d and G82-j, MIC > 50 μg·ml1; G82-h,
G82-i and G82-k, MIC = 20 μg·ml1) were significantly
higher than the MIC of G82 (3 - 5 μg·ml1) (Table 3).
4. Discussion
The work herein discussed made a partial sequence of the
gene of E. necator available; it will be helpful for further
Observation on Baseline Sensitivity of Erysiphe necator Genetic Groups to Azoxystrobin 1643
300 bp
A: St I
B: St II
Figure 1. Electrophoretic profiles obtained by amplification of E. necator DNA with the primers RSCBF1-RSCBR2, and se-
quences of St I (A) and St II (B).
Erysiphe necator (StI)
Erysiphe graminis (sensitive)
Erysiphe graminis (resistant)
Magnaporthe grisea
Saccharomyces cerevisiae
Venturia inaequalis
Podosphaera fusca (sensitive)
Podosphaera fusca (resistant)
Amino acid position
:FILMMATAFLGYVLPYGQMSLWGATVITNLMSAIPWIGQDIV
:FILMIVTAFLGYVLPYGHMSHWGATVITNLMSAIPWIGQDIV
:FILMIVTAFLGYVLPYGHMSHWAATVITNLMSAIPWIGQDIV
:LILMMAIGFLGYVLPYGQMSLWGATVITNLISAIPWIGQDIV
:FTLTIATAFLGYCCVYGQMSHWGATVITNLFSAIPFVGNDIV
:FILMIVTAFLGYVLPYGQMSLWGATVITNLMSAIPWIGQDIV
:--------FLGYGLPYGQMSLWGATV----------------
:--------FMGYGLPWGQMSLWAATV----------------
* * ** * ** * ******* **** * ***
:121 129 143 162
Figure 2. StI translation nucleic acid to amino acid (underlined) and multiple alignments with the cytochrome b gene frag-
ments containing the point mutations responsible of resistance to QoI-STARs in other fungi (in bold, position 129 and 143).
Table 2. Putative mutants of E. necat o r resistant to azoxystrobin obtained by selection of spontaneous mutations.
Colony transfer at 1-month intervals and fungicide concentration (μg·ml1)
Starting strain
1st 2nd 3rd 4th 5th 6th
Recovered strains
5 G82-a
1 5 G82-b
5 10 G82-c
1 1 G82-d
1 10 G82-e
1 1 10 G82-f
1 1 G82-g
1 10 G82-h
1 1 10 100 G82-i
1 1 0 20 G82-j
1 10 20 G82-k
G82 0.1 1
1 1 10 30 G82-l
Copyright © 2012 SciRes. AJPS
Observation on Baseline Sensitivity of Erysiphe necator Genetic Groups to Azoxystrobin
1644
Table 3. Putative resistant mutants response to azoxystrobin.
Diameter (mm) of 14-days colony
Fungicide
concentration (μg·ml1) G82 G82-a G82-d G82-h G82-i G82-j G82-k
0 11 12 9 11 4 5 12
0.1 5 5 4 3 3 3 5
0.3 4 4 3 - 3 4 2
1 3 4 2 2 4 2 -
5 0 0 3 - 3 0 2
10 0 0 3 2 4 1 1
20 0 0 0 0 0 0 0
50 0 0 3 0 0 2 0
EC50 <0.1 <0.1 <0.1 <0.1 10 - 20 0.3 - 1 <0.1
MIC 5 5 >50 20 20 >50 20
researches aiming at developing molecular methods use-
ful for field monitoring of the fungal resistance to fungi-
cides.
Attempts of obtaining laboratory mutants resistant to
QoI-STAR fungicides were only partially successful,
since only few mutants were obtained and usually they
displayed a low resistance level. Based on information
known in other fungi, mitochondrial resistance may be
unstable, since mitochondrial genome is present at a rela-
tively high number of copies, and it is very unlikely that
all of them carry resistance mutations [17].
An in vitro technique allowed to establish baseline
sensitivity of E. necator to azoxystrobin fungicide were
optimized. No significant differences in response to the
fungicides were observed among the tested isolates.
Nevertheless, differences were detected between group
A and group B isolates. The same behavior of the two
groups was observed in early study in response to triadi-
menol (DMIs) Fungicide [2].
The higher sensitivity to azoxystrobin of group A iso-
lates might play a role in their disappearance in vineyards
from June onwards, when intensive fungicides treatments,
including azoxystrobin, are routinely applied. Type A
isolates are usually collected on typical flag shoots early
in April, at the time of highest susceptibility of shoots to
powdery mildew, corresponding to the phenological
stage BBCH 13 - 16 (i.e. three to six unfolded leaves) [17,
18]. At this time, type A isolates have a higher chance to
colonise the forming buds in which they can remain until
the next season. This was further confirmed by the fact
that the incidence of flag shoots in a vineyard can be
predicted by assessing the extent of bud infection in the
preceding year [19]. The overwintering mode of group A
isolates determines their prevalent asexual reproduction.
This seems to be confirmed also by the low genotypic
diversity found in this group, and by the unbalanced dis-
tribution of the two mating type alleles that characterizes
this group [1,20,21]. If isolates of group A are present
only at the beginning of the growing season, they have a
reduced opportunity to be exposed to the fungicide pres-
sure. This fact, coupled with the absence of the gene re-
arrangement consequent to sexual reproduction, would
make group A isolates less likely to develop resistance.
In contrast, group B isolates develops later in the sea-
son and reproduce mostly sexually [22]. Thus, recombi-
nants would have a better opportunity of pyramiding
mutations necessary to build the fungicides resistance
that these isolates show, and that enables them to play a
primary role in epidemics.
Further studies are required for a better understanding
of E. necator resistance to azoxystrobin and for clarify-
ing the complex interactions that this trait appears to
have with population genetics of this pathogen. Ascer-
taining if the differential sensitivity of isolates of the two
genetic groups extends also to other fungicide groups,
identifying other factors that may favor one group over
the other, and understanding how all these factors can
affect the evolution of the epidemics, will be of great
help for a more efficient disease control.
5. Acknowledgements
The Author would like to thank Prof. Franco Faretra and
Dr. Monica Miazzi from Faculty of Plant Protection and
Microbiological Application, Bari University, for their
generous support and facilitating carrying out this work.
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