SENSITIVITY OF Venturia inaequalis ISOLATES TO ... - Izbis

SENSITIVITY OF Venturia inaequalis ISOLATES TO
FUNGICIDES WITH DIFFERENT MODES OF ACTION
Goran Aleksić 1 , Tatjana Popović 1 , Mira Starović 1 , Slobodan Kuzmanović 1 ,
Dragana Jošić 2 , Nenad Dolovac 1 , Dobrivoj Poštić 1
1
Institute for Plant Protection and Environment, Belgrade,Serbia
2
Institute for Soil Science, Belgrade,Serbia

Apple scab caused by Venturia inaequalis (Cooke)
Winter (anamorph Spilocaea pomi Fr.) is the most
important apple disease in the world. Chemical
control with fungicides has been the main measure of
disease control. During the growing season, in order
to keep the trees scab free, more than 20 treatments
may be needed. In order to protect apple trees from
the apple scab, a wide spectrum of different chemical
preparations is used from different chemical groups,
with different mode of action (preventative,
preventative-curative, curative and eradicative) which
are often combined in order to enhance their
effectiveness or avoid development of resistance.

Unfortunately, fungicide resistance has been
observed in some orchards. Investigating
occurrence of reduced sensitivity of pathogens to
fungicides is of great importance in conditions of
intensive crop protection.

Fungicide resistance in V. inaequalis is well
documented worldwide for old fungicides such
as dodine (Köller and Wilcox, 1999; Broniarek-
Niemiec and Bielenin, 2008), benzimidazole
(Koenraadt et al., 1992), demethylation inhibitors
(DMIs) (Sholberg and Haag, 1993; Köller et al.,
1997), and newer fungicides such as strobilurins
(Olaya and Koller, 1999; Fontaine et al., 2009) and
anilinopyrimidines (Küng et al., 1999). There are
no reports in the literature about the sensitivity to
mancozeb, an old fungicide still used for apple
scab control, either used alone or mixed mainly
with DMIs as an anti-resistance strategy.

The objective of this research was to determine
the sensitivity of V.inaequalis populations
isolated from treated and untreated apple
orchards to the difenoconazole, flusilazole,
cyprodinil, pyrimethanil, kresoxim-methyl,
captan and mancozeb fungicides.

MATERIAL AND METHODS
The purpose of this trial is to test, in vitro, the
effectiveness of some fungicides on the mycelia
growth of Venturia inaequalis isolated from
infected apple leaves collected from treated
(isolate Morovic – M and M1) and untreated
(isolate Nestin – N and N1) orchards during 2011.
Isolations were carried out according to the
method of Borić (1987) and Aleksić (1996).
Isolates were grown for six weeks on PDA at
20°C.

Commercial formulations of seven fungicides,
representing different chemical families, were
used in this study. The experiment was done
using a method described by Popović (2004).
Appropriate volumes of each fungicide were
added to the PDA at approximately 50°C in the
quantities needed to achieve the final
concentrations at the rate of the registered dose
and poured in sterilized 50 mm Petri plates.

Active
ingredient
TABLE 1. Fungicides on tested fungal cultures
of Venturia inaequalis
Fungicide
Tested
concentrations
Tested
doses / litar
Difenoconazole Score 250 EC 0.02 1 (0.005) 2 0.2 mL
Flusilazole Punch 40 EC 0.01 1 (0.004) 2 0.1 mL
Cyprodinil Chorus 75 WG 0.025 1 (0.01875) 2 0.25 g
Pyrimethanil Pyrus 400 SC 0.15 1 (0.06) 2 1.5 mL
Kresoximmethyl
Stroby DF 0.02 1 (0.01) 2 0.2 g
Captan Merpan 80 0.2 1 (0.16) 2 2.0 g
Mancozeb Mankogal 0.25 1 (0.2) 2 2.5 g
1
% of fungicide
2
% of active ingredient

Mycelia plug (1 mm in diameter), obtained from the
actively growing cultures, were transferred to
fungicide amended plates. The Control was PDA
plates without the addition of the fungicide. There
were four replicates for each fungicide. The dishes
were incubated at 20°C in the dark and the diameter
of each colony was measured twice perpendicularly.

RESULTS
Four isolates (M, M1, N1 and N) of V. inaequalis,
originating from two localities on the teritory of Serbia
(Morović and Neštin) were used for investigations in this
work. Isolates from Morović locality were sampled from
commercial apple orchards area of 100 ha, in which
intensive measures for apple scab control are
implementing for many years. This orchard had
applications of the entire range of fungicides registered
for apple scab control. Isolates from locality Neštin were
taken from individual apple trees distant from any
commercial orchards. No fungicides have been applied
for apple scab control, and these isolates could be
considered wild or organic isolates.

TABLE 2. Growth of four Venturia inaequalis isolates
on PDA medium with tested fungicides
Active
ingredient
Fungicide
Dose/
lit.
Isolate (average growth in mm)
M D.t. M1 D.t. N D.t. N1 D.t.
Difenoconazole Score 250EC 0.2 mL 1.0 a 1.0 a 1.0 a 1.0 a
Flusilazole Punch 40 EC 0.1 mL 1.0 a 1.0 a 1.0 a 1.0 a
Cyprodinil Chorus75WG 0.25 g 1.0 a 1.0 a 1.0 a 1.0 a
Pyrimethanil Pyrus 400SC 1.5 mL 1.0 a 1.0 a 1.0 a 1.0 a
Kresoximmethyl
Stroby DF 0.2 g 1.0 a 12.75 b 1.0 a 1.0 a
Captan Merpan 80 2.0 g 1.0 a 1.0 a 1.0 a 1.0 a
Mancozeb Mankogal 2.5 g 1.0 a 1.0 a 1.0 a 1.0 a
Control - - 19.8 b 20.13 c 21.88 b 29.38 b
LSD 0.05
0.44 0.61 2.82 0.33

Growth in mm
Growth of four Venturia inaequalis isolates on PDA medium
with tested fungicides
30
25
20
15
10
5
Difenoc.
Flusil.
Cyprod.
Pyrimet.
Kr.-methyl
Captan
Mancozeb
Control
0
M M1 N N1
Isolates

FIGURE 1. Isolate M; K-control, D-difenoconazole, F-flusilazole,
P-pyrimethanil, C-cyprodinil, S-kresoxim-methyl

FIGURE 2. Isolate M; K-control, D-difenoconazole, F-flusilazole,
C-cyprodinil, P-pyrimethanil, S-kresoxim-methyl

DISCUSSION
V. inaequalis is the most important disease in apple
production. About 75% of the total application of
pesticides applied in the production of apples are for the
fungal diseases control (of which 70% is used for apple
scab control alone. A major problem in protecting apples
from this pathogen is the fact that a small number of
fungicides are being used in ever increased quantity. This
fact contributes to the occurence of pathogen resistance
or reduced susceptibility of the pathogen towards used
fungicides, due to capacity to the pathogen for genetic
variation (Aleksić et al., 2005). According to Köller (1988)
most fungicides used to control pathogens have a
specific mechanism of action (eg. DMI fungicides), and it
was clear that there is a risk of resistance development.

According to the results of testing the biological efficacy of
some fungicides (QoI and DMI), conducted during two growing
periods, Aleksić (2006) found that the examined products
showed high efficiency in both preventive and curative control
of apple scab. Similar results have been announced Balaz and
Knezevic (2003) examining the efficacy of newer fungicides in
controlling apple scab and apple powdery mildew. In studies
conducted during 1998, 1999 and 2002, the authors found that
the best efficacy in controlling apple scab was demonstrated
by a group of strobilurins (trifloxystrobin and kresoxim-methyl)
They found no statistically significant differences in the
efficiency of the the preparations tested in this group of
compounds. They then tested a group of DMI fungicides -
Difenoconazole (product-Score 250 EC), which exhibited a
lower efficacy in controlling apple scab.
In our in vitro experiments, colony growth of V. inaequalis was
registered only in the treatment with kresoxim-methyl, which
leads to the conclusion that there was an appearance of
pathogens resistance to this fungicide.

Recent studies by other authors identified reduced
sensitivity of pathogen to the compounds of the
strobilurin group (Köller et al., 2004) which is consistent
with the results obtained in this paper. According to Kuck
and Mehl (2003), the resistance to strobilurins in the field
is noticed on several pathogens since 1998. Mutations of
the G143A are mostly responsible for the occurence of
this resistance. According to Brunelli et al. (2003), in the
case of fungicides in the strobilurin group, the second
generation fungicides (trifloxystrobin) exhibited greater
efficacy than the previous generation (kresoksim-methyl).

The occurence of resistance in another very important
group of fungicides – DMI, was observed even twenty
years ago. The problems with decreasing effectiveness of
these fungicides can occur after several years of their
intensive use. Among them there is no cross-resistance.
The genetic basis of resistance to the DMI is complex and
has not been sufficiently studied. It is polygenic and
developed through a process of gradual decline in
pathogen sensitivity to fungicides (Gaunt et al., 1996).
According to Pscheidt (2004) resistance to DMI fungicides
in the United States is noticed in several states and in
several cultures. The powdery mildew and apple scab
causal agents are the first pathogens in which the
emergence of resistance observed.