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Fungicide sensitivity of Trichoderma spp. from Agaricus

bisporus farms in Serbia
a b c c b
Dejana Kosanović , Ivana Potočnik , Jelena Vukojević , Mirjana Stajić , Emil Rekanović ,
b b
Miloš Stepanović & Biljana Todorović
a
Institute of Virology, Vaccines and Sera “Torlak,” Belgrade, Serbia
b
Institute of Pesticides and Environmental Protection, Belgrade-Zemun, Serbia
c
Institute of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia
Published online: 11 Jun 2015.

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To cite this article: Dejana Kosanović, Ivana Potočnik, Jelena Vukojević, Mirjana Stajić, Emil Rekanović, Miloš Stepanović
& Biljana Todorović (2015) Fungicide sensitivity of Trichoderma spp. from Agaricus bisporus farms in Serbia, Journal of
Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 50:8, 607-613, DOI:
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 Journal of Environmental Science and Health, Part B (2015) 50, 607–613
Copyright © Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601234.2015.1028849

Fungicide sensitivity of Trichoderma spp. from Agaricus

bisporus farms in Serbia

DEJANA KOSANOVIC
1, IVANA POTOCNIK

 2
3, MIRJANA STAJIC
, JELENA VUKOJEVIC
3,

2 
2
EMIL REKANOVIC , MILOS STEPANOVIC and BILJANA TODOROVIC
2

1
Institute of Virology, Vaccines and Sera “Torlak,” Belgrade, Serbia
2
Institute of Pesticides and Environmental Protection, Belgrade-Zemun, Serbia
3
Institute of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia
Downloaded by [FU Berlin] at 15:43 24 June 2015

Trichoderma species, the causal agents of green mould disease, induce great losses in Agaricus bisporus farms. Fungicides are widely
used to control mushroom diseases although green mould control is encumbered with difficulties. The aims of this study were,
therefore, to research in vitro toxicity of several commercial fungicides to Trichoderma isolates originating from Serbian and Bosnia-
Herzegovina farms, and to evaluate the effects of pH and light on their growth. The majority of isolates demonstrated optimal
growth at pH 5.0, and the rest at pH 6.0. A few isolates also grew well at pH 7. The weakest mycelial growth was noted at pH 8.0–
9.0. Generally, light had an inhibitory effect on the growth of tested isolates. The isolates showed the highest susceptibility to
chlorothalonil and carbendazim (ED50 less than 1 mg L¡1), and were less sensitive to iprodione (ED50 ranged 0.84–6.72 mg L¡1),
weakly resistant to thiophanate-methyl (ED50 D 3.75–24.13 mg L¡1), and resistant to trifloxystrobin (ED50 D 10.25–178.23 mg
L¡1). Considering the toxicity of fungicides to A. bisporus, carbendazim showed the best selective toxicity (0.02), iprodione and
chlorothalonil moderate (0.16), and thiophanate-methyl the lowest (1.24), while trifloxystrobin toxicity to A. bisporus was not tested
because of its inefficiency against Trichoderma isolates.
Keywords: Green mould disease, antifungal activity, cultivated mushroom.

Introduction T. aggressivum accounted for mushroom yield losses of

World production of button mushrooms is over 3 million
tons annually. European countries account for 50% of the
overall world production, of which 87% belong to EU
countries and 13% to countries outside the EU.[1] The
most important fungal pathogens of edible mushrooms are
Trichoderma species, such as Trichoderma harzianum, Tri-
choderma atroviride, Trichoderma koningii, Trichoderma
virens, etc., which are present in mushroom beds and have
different abilities to cause disease.[2] In the 1990s, T.
aggressivum appeared and reached epidemic proportions.
Unlike other Trichoderma species whose mycelium formed
concentric rings after sporulation, the isolates of T. aggres-
sivum f. europium formed white pustules, and had larger
conidia and faster growth rate than T. harzianum. A phy-
logenetic analysis showed that T. harzianum was most
closely related to the aggressive mushroom colonizer T.
aggressivum f. europaeum.[2] Green mould caused by
Address correspondence to Ivana Potocnik, Institute of Pesti-
cides and Environmental Protection, Banatska 31b, Belgrade
11080, Serbia; E-mail: ivana.potocnik@pesting.org.rs
Received January 2, 2015.
between 60% and 100%.[3,4] It first appeared in the British
Isles (T. aggressivum f. europaeum) and North America
(T. aggressivum f. aggressivum), and then spread into
mushroom farms all over Western Europe over the next
few years.[3,5–10] During the past decade, the pathogen has
dispersed into Central and South East Europe, Central
America and Australia.[2,11–15] Green mould caused by
Trichoderma spp. is characterized by white mycelia of fast-
growing colonies on casing or substrate that change color
to green after profuse sporulation. Spots on fruiting bodies
appear in the advanced stage of disease, while basidiocarps
fail to form during a serious outbreak.[3]
Disease control in mushroom farms worldwide includes
application of only a few fungicides. Prochloraz manga-
nese has been officially recommended in mushroom indus-
try in Europe, and chlorothalonil, thiabendazol and
thiophanate-methyl in North America.[16,17] However,
there is still no solution for the green mould disease on
mushrooms. No fungicide has been registered for use on
mushrooms in Serbia, but Serbian growers nevertheless
frequently apply fungicides registered for other crops, such
as carbendazim, thiophanate-methyl and prochloraz man-
ganese.[18] In a previous study, prochloraz manganese and
Bacillus subtilis were found to be highly toxic to Serbian
 608 Kosanovi

c et al.

Trichoderma isolates, while tea tree oil did not exhibit a
significant antifungal activity.[2] The survey continued on
five more fungicides: carbendazim, thiophanate-methyl,
chlorothalonil, iprodione and trifloxystrobin, which have
already been used against other fungal pathogens on
mushrooms before: Mycogone perniciosa, Lecanicillium
(Verticillium) fungicola and Cladobotryum sp.[18–22] Thia-
bendazole, which is used in mushroom production world-
wide, was not included in the survey because it is not
available for use in Serbia.[18] It is very important to deter-
mine optimum conditions for pathogen growth, such as
pH and light intensity, in order to improve disease control.
The aim of this study was to examine in vitro toxicity of
several fungicides to Trichoderma isolates obtained from
A. bisporus farms in Serbia and Bosnia-Herzegovina, com-
pared to prochloraz. The impacts of pH and light on the
growth of Trichoderma isolates were also evaluated.
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them considering their morphology and mycelium growth.
Also, they were different from all known Trichoderma spe-
cies.[2] The isolates have been maintained on potato dex-
trose agar (PDA) at 5 C in the culture collection of the
Institute of Pesticides and Environmental Protection,
Belgrade.
Selected pH values and light intensity
Trichoderma isolates were grown at 20 C on PDA (agar,
Torlak, Belgrade, Serbia; dextrose, Torlak, Belgrade, Ser-
bia; peeled potato). The medium was inoculated with
mycelium agar discs (Ø 10 mm) taken from the edge of
4 day-old cultures of each tested isolate and incubated for
72 h. The effect of pH, adjusted by 0.1 M HCl or 0.1 M
NaOH before sterilization, was studied over a range from
5.0 to 9.0. To examine the effect of light effect, the isolates

Materials and methods were grown under constant neon light (λ D 533–725 nm)
or in the dark. Six replicates were made per isolate and
Isolates and growth conditions treatment.

Twenty Trichoderma isolates were collected from 13 Ser-

bian A. bisporus farms and one in Bosnia-Herzegovina
during 2006–2010 and were identified to species level in a
previous study by standard mycological studies and ITS1/
ITS4 sequence analyses (Table 1).[2] There were three sepa-
rate groups that were not identified to species level. They
were most similar to T. harzianum isolates as molecular
analysis proved their relatedness, but distinguishable from
Fungicides
Five commercial fungicides: carbendazim, chlorothalonil,
iprodione, thiophanate-methyl and trifloxystrobin
(Table 2) were tested as potential antifungal agents against
the studied. Trichoderma isolates.

Table 1. Tested Trichoderma spp. isolates and their origin.

Year of
Species Code Origin Substrate collection
Trichoderma aggressivum f. T76 Lisovi
ci, Barajevo, Serbia Mushroom substrate 2010
europaeum Samuels and W. Gams T77 Lisovi
ci, Barajevo, Serbia Mushroom substrate 2010
T85 Lisovi
ci, Barajevo, Serbia Mushroom substrate 2010
Trichoderma atroviride P. Karst. T33 Zemun Polje, Serbia Fruiting body 2006
T60 Zemun Polje, Serbia Fruiting body 2008
Trichoderma harzianum Rifai T10 Pozarevac, Serbia Fruiting body 2006
T52 Institute of Pesticides, Belgrade, Serbia Fruiting body 2008
T54 Kula, Serbia Fruiting body 2008
T64 Institute Zemun, Serbia Fruiting body 2009
T91 Institute Zemun, Serbia Fruiting body 2010
Trichoderma koningii Oudem. T39 Veliko Gradiste, Serbia Fruiting body 2007
Trichoderma virens (J.H.Mill., T5 Komiri
c, Serbia Fruiting body 2006
Giddens & A.A.Foster) Arx
Trichoderma sp. group 1 T1 Komiri
c, Serbia Fruiting body 2006
Trichoderma sp. group 2 T63 Becej, Serbia Fruiting body 2009
Trichoderma sp. group 3 T37 Novi Slankamen, Serbia Fruiting body 2007
T69 Sarajevo, Bosnia and Herzegovina Fruiting body 2009
T71 Mol, Serbia Fruiting body 2010
T86 Skobalj, Serbia Fruiting body 2010
T88 Veliko Gradiste, Serbia Fruiting body 2010
T90 Zemun Polje, Serbia Fruiting body 2010
 Fungicides against Trichoderma spp. on mushrooms 609

Table 2. Characteristics of selected fungicides.

Active
ingredient (a.i.) Carbendazim Chlorothalonil Iprodione Thiophanate-methyl Trifloxystrobin
Trade name Galofungin WP Bravo 750 SC Kidan EC Funomil WP Zato50 WG
Formulation 500 g kg¡1 720 g L¡1 260 g L¡1 700 g kg¡1 500 g kg¡1
Supplier Galenika– Syngenta Bayer Crop Agromarket Bayer Crop
Fitofarmacija Science Science
Chemical Class Benzimidazole Chloronitril Dicarboximide Benzimidazole Strobilurin

Antifungal activity isolate were used to enable statistical analysis. Fungicide

concentrations inhibiting mycelial growth by 50% (ED50)
Activity of the selected fungicides against Trichoderma
were determined for each isolate. These values were defined
isolates were tested by macrodilution method on PDA
for each isolate by interpolation from computer-generated
(pH D 7) amended with each tested fungicide, ranging
log-probit plots of fungicide concentration and relative inhi-
from 0.01 to 1000.00 mg L¡1.[23] Based on earlier observa-
bition.[24] Fungicide effects were studied by analyzing the
tion, fungicide concentrations selected for the study
means and variance of ED50 by the multiple range test.[25]
included: iprodione 0.47, 0.94, 1.87 and 3.75 mg L¡1,
The isolates were considered resistant to a fungicide if its
chlorothalonil and carbendazim 0.19, 0.37, 0.75 and
ED50 exceeded 50.0 mg L¡1, weakly resistant if the ED50
1.50 mg L¡1, thiophanate-methyl 6.25, 12.50, 25.00 and
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was in the range between 5.0 and 50.0 mg L¡1, and sensi-
50.00 mg L¡1, and trifloxystrobin 0.10, 1.00, 10.00
tive if the value was less than 5.0 mg L¡1.[26] A selectivity
and 100.00 mg L¡1.[18] The selected fungicide volumes
index for each active ingredient was calculated as a ratio of
were added to molten sterile medium (50 C). The fungi-
the ED50 mean value for each tested isolate of the pathogen
cide-amended media and fungicide-free media, used as the
and a corresponding estimate for A. bisporus.[19]
control were inoculated with mycelium agar discs
(Ø 10 mm) taken from the edge of 4 day-old cultures of
Trichoderma isolates and incubated at 20 C. Colony diam-
eter was measured after 72 h. Mycelial growth on the Statistical analyses
fungicide-amended media was measured as percentage Data were examined by one-way analysis of variance,
against the control. Three replicates per treatment and including a comparison of means by F-test. The test was

Table 3. Effect of pH on mycelial growth of Trichoderma spp. isolates.

Colony diameter after 72-h-old cultivation (mm § SE)

Species Code pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0

Trichoderma aggressivum f. europaeum T76 36.7 § 0.4 B
39.2 § 0.5 A
37.5 § 0.2 B
34.8 § 0.5 C
34.3 § 0.7C
T77 36.3 § 0.3A 32.8 § 0.7B 33.3 § 0.3B 31.0 § 0.6C 30.8 § 0.8C
T85 38.5 § 1.1A 36.5 § 0.7AB 37.0 § 0.4A 34.7 § 0.8B 34.3 § 0.6C
Trichoderma atroviride T33 41.2 § 1.0A 40.8 § 0.6A 38.3 § 2.0AB 38.0 § 0.9AB 33.1 § 1.1B
T60 35.0 § 0.4AB 37.0 § 0.0A 36.5 § 0.7A 33.3 § 1.3BC 32.2 § 1.0C
Trichoderma harzianum T10 42.0 § 0.9A 39.0 § 0.4B 39.3 § 0.4AB 36.0 § 1.4C 35.2 § 1.1C
T52 36.8 § 0.6A 33.7 § 0.2B 33.5 § 1.5B 32.8 § 1.4B 32.5 § 0.5B
T54 32.2 § 0.6A 29.2 § 0.7B 28.8 § 0.7BC 27.5 § 0.2C 24.8 § 0.4D
T64 38.5 § 0.8A 36.5 § 1.1B 36.8 § 0.4AB 35.2 § 0.4BC 33.7 § 0.5C
T91 32.3 § 0.3A 32.3 § 1.0A 32.2 § 1.4AB 29.8 § 0.6BC 27.7 § 0.2C
Trichoderma koningii T39 43.5 § 0.5AB 44.3 § 0.3A 43.2 § 0.3AB 42.3 § 0.6B 38.0 § 1.1C
Trichoderma virens T5 30.7 § 0.4A 29.5 § 0.6AB 28.0 § 1.0BC 27.5 § 0.2C 26.7 § 0.2C
Trichoderma sp. Group 1 T1 15.7 § 0.4A 14.5 § 0.3B 14.3 § 0.2B 14.7 § 0.2B 14.0 § 0.0B
Trichoderma sp. Group 2 T63 29.5 § 0.8BC 32.7 § 0.4A 30.2 § 0.8B 30.0 § 0.4B 28.3 § 0.2C
Trichoderma sp. Group 3 T37 37.7 § 0.3B 38.8 § 0.4A 36.5 § 0.4C 35.7 § 0.5C 34.5 § 0.2D
T69 34.3 § 1.6AB 34.7 § 0.2AB 35.5 § 0.3A 32.7 § 0.4BC 30.7 § 1.1C
T71 35.2 § 0.7C 37.5 § 0.6AB 38.5 § 1.0A 36.0 § 0.9BC 35.8 § 0.3BC
T86 35.8 § 1.0A 36.3 § 0.3A 37.0 § 0.7A 34.8 § 0.5A 31.5 § 1.3B
T88 42.3 § 0.6A 39.5 § 0.8B 38.3 § 0.3BC 37.5 § 0.3C 34.3 § 0.6D
T90 36.2 § 0.9A 35.7 § 0.3A 35.0 § 0.4AB 33.2 § 0.5BC 31.5 § 0.5C
Means in row followed by the same uppercase letter are not significantly different at P < 0.05.
 610 Kosanovi

c et al.

used to compare average colony diameters of each tested
isolate under the influence of different pH values and
light. In all analyses, the level of significance was at least
P < 0.05.[27] Statistical data analysis was performed using
the software Statistica for Windows 6.0 (Stat Soft Italia,
1997).
Results
light (Table 4). Statistically significant (P < 0.05) increases
in colony diameters were found for T. atroviride, T.
aggressivum f. europaeum, T. harzianum, and Trichoderma
sp. group 3 isolates. T. harzianum T10 showed the maxi-
mum growth both under neon light and in the dark (52.0
and 54.0 mm, respectively). Trichoderma sp. group 1 T1
had only half of the colony diameter of the other isolates
after 72 h of cultivation both under illumination and in
the dark (17.3 and 18.0 mm, respectively).

Effects of pH and light on isolate growth

Twelve tested isolates grew best under pH 5.0, five isolates
under pH 6.0 and three under pH 7.0. Medium pH 5.0, 6.0
and 7.0 showed the same effect on the growth of T. harzia-
num T91, while the lowest growth of all isolates was
observed under pH 9.0 (Table 3). Declining growth rate
with increasing pH value was noted in 12 isolates, while its
maximum was reported at pH 6.0 or 7.0 in the remaining
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Sensitivity of Trichoderma isolates to fungicides
The tested Trichoderma isolates showed different levels of
sensitivity to the selected fungicides in vitro (Table 5).
Chlorothalonil and carbendazim concentrations below
0.19 mg L¡1 failed to affect growth, while concentrations
of 0.75 mg L¡1 and higher inhibited the growth of most
isolates severely. The respective ED50 values of chlorotha-

8, which was particularly evident for the isolates of Tricho-
derma sp. group 3. Growth exceeding 40.0 mm after 72 h
was observed in T. atroviride T33, T. harzianum T10, Tri-
choderma sp. group 3 T88 at pH 5.0, while a maximum
was reached by T. koningii T39 (44.3 mm) under pH 6.0.
Trichoderma sp. group 1 T1 was characterized by a signifi-
cantly lower colony diameter, regardless of pH value,
ranging between 15.7 and 14.0 mm, which is the lowest
growth observed (Table 3).
The data show faster growth of all tested Trichoderma
isolates in the dark, compared to their growth under neon
lonil and carbendazim were 0.12–0.97 and 0.25–0.92 mg
L¡1. Most Trichoderma isolates were capable to grow at
0.47 mg L¡1 iprodione concentration but growth was
inhibited by 3.75 mg L¡1 and higher concentrations. The
values of iprodione ED50 for Trichoderma isolates were in
a range of 0.84–6.72 mg L¡1. Growth of the isolates was
good at thiophanate-methyl concentration 6.25 mg L¡1
and trifloxystrobin concentration lower than 10 mg L¡1.
Severely inhibited growth of Trichoderma isolates was
observed at thiophanate-methyl concentration of 25 mg
L¡1 and trifloxystrobin concentration of 100 mg L¡1.

Table 4. Effect of light intensity on mycelial growth of Trichoderma sp. isolates.

Colony diameter after 72 h incubation (mm § SE)

Species Code Light Dark

Trichoderma aggressivum f. europaeum T76 42.7 § 0.7 A
43.3 § 0.6A
T77 36.3 § 0.3A 37.8 § 0.2B
T85 43.2 § 0.7A 46.2 § 1.0B
Trichoderma atroviride T33 29.5 § 0.2A 40.3 § 1.4B
T60 44.8 § 1.0A 47.0 § 1.3A
Trichoderma harzianum T10 52.0 § 0.9A 54.0 § 0.0B
T52 43.3 § 0.2A 44.0 § 0.0B
T54 40.3 § 0.2A 42.0 § 0.0B
T64 38.0 § 0.0A 40.5 § 0.2B
T91 36.2 § 0.2A 37.0 § 0.2B
Trichoderma koningii T39 38.7 § 0.4A 39.0 § 0.4A
Trichoderma virens T5 33.0 § 0.8A 34.0 § 0.0A
Trichoderma sp. group 1 T1 17.3 § 0.7A 18.0 § 0.0A
Trichoderma sp. group 2 T63 41.0 § 1.3A 42.8 § 1.0A
Trichoderma sp. group 3 T37 39.0 § 0.0A 40.5 § 0.2B
T69 48.7 § 0.4A 50.5 § 0.3B
T71 34.0 § 0.0A 41.5 § 0.7B
T86 44.0 § 1.8A 49.7 § 0.2B
T88 42.3 § 0.5A 42.8 § 0.2A
T90 35.0 § 0.0A 43.5 § 0.2B
Means in row followed by the same uppercase letter are not significantly different at P < 0.05.
 Fungicides against Trichoderma spp. on mushrooms 611

Table 5. Effect of tested fungicides on the growth of Trichoderma spp. isolate.

ED50 (mg L¡1) mean and range

Species Code Carbendazim Chlorothalonil Iprodione Thiophanate-methyl Trifloxystrobin

Trichoderma T76 0.61 (0.54–0.68) 0.33 (0.27–0.41) 1.76 (1.29–2.56) 18.92 (16.71–21.11) 10.62 (5.56–22.48)
aggressivum f. T77 0.31 (0.28–0.34) 0.97 (0.80–1.26) 0.96 (0.76–1.26) 13.43 (12.05–15.11) 10.25 (4.10–65.69)
europaeum T85 0.60 (0.51–0.76) 0.45 (0.31–0.90) 3.04 (2.09–5.58) 13.75 (11.31–16.36) 84.55 (49.41–177.54)
Trichoderma T33 0.35 (0.28–0.42) 0.25 (0.19–1.30) 6.08 (3.21–22.63) 3.75 (2.49–4.88) 97.41 (21.90–4348.42)
atroviride T60 0.25 (0.22–0. 82) 0.14 (0.05–0.23) 1.29 (0.97–1.90) 5.28 (3.40–7.02) 17.57 (7.65–55.96)
Trichoderma T10 0.70 (0.62–0.78) 0.48 (0.38–0.61) 1.57 (1.29–1.98) 11.91 (9.84–16.00) 36.89 (16.86–113.50)
harzianum T52 0.35 (0.32–0.39) 0.22 (0.13–0.33) 2.10 (1.71–2.77) 7.24 (6.23–8.45) 151.02 (25.07–34070.19)
T54 0.30 (0.26–0.33) 0.12 (0.08–0.15) 1.36 (0.93–2.43) 24.13 (15.67–69.94) 34.78 (20.56–100.92)
T64 0.33 (0.27–0.39) 0.33 (0.29–0.38) 1.29 (1.01–1.78) 6.86 (5.83–7.92) 21.62 (5.15–465.45)
T91 0.37 (0.33–0.42) 0.21 (0.17–0.25) 1.16 (0.58–4.75) 9.29 (8.03–10.87) 34.28 (10.56–660.28)
Trichoderma koningii T39 0.92 (0.79–1.06) 0.97 (0.81–1.22) 2.98 (2.03–5.25) 8.74 (7.13–10.48) 21.07 (11.91–43.34)
Trichoderma virens T5 0.34 (0.31–0.39) 0.31 (0.26–0.36) 2.03 (1.54–2.99) 6.78 (6.02–7.69) 34.18 (8.97–1144.64)
Trichoderma sp. T1 0.28 (0.24–0.32) 0.50 (0.40–0.70) 1.27 (1.02–1.65) 5.67 (4.68–6.66) 178.23 (67.59–938.73)
group 1
Trichoderma sp. T63 0.41 (0.37–0.46) 0.36 (0.26–0.75) 6.72 (3.62–20.92) 19.02 (14.82–25.50) 52.87 (19.50–413.60)
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group 2
Trichoderma sp. T37 0.30 (0.26–0.34) 0.71 (0.56–0.92) 3.08 (1.67–12.18) 20.00 (15.88–27.40) 33.67 (8.93–1061.95)
group 3 T69 0.32 (0.28–0.36) 0.23 (0.19–0.28) 1.03 (0.81–1.34) 21.67 (17.77–25.07) 21.47 (9.36–68.14)
T71 0.27 (0.23–0.31) 0.31 (0.25–0.38) 2.20 (1.96–2.54) 11.06 (9.85–12.39) 65.80 (29.14–238.27)
T86 0.31 (0.28–0.35) 0.22 (0.17–0.27) 0.89 (0.71–1.12) 10.58 (9.43–11.84) 25.74 (9.31–203.87)
T88 0.44 (0.40–0.48) 0.32 (0.24–0.43) 1.14 (0.83–1.70) 17.43 (15.63–19.43) 22.69 (5.91–1091.50)
T90 0.40 (0.35–0.46) 0.40 (0.34–0.47) 0.84 (0.75–1.08) 12.90 (10.46–16.68) 51.67 (18.37–298.37)
95% confidence interval (P D 0.05).

Thiophanate-methyl ED50 values ranged 3.75–24.13 mg
L¡1, and those of trifloxystrobin 10.25–178.23 mg L¡1.
Based on the presented data, medial ED50 values were cal-
culated for all tested isolates per fungicide (carbendazim
0.41; chlorothalonil 0.39; iprodione 2.14; thiophanate-
methyl 12.42) in order to define the selective toxicity of
fungicides to the pathogen and host (A. bisporus F56).
Discussion
The results indicate that pH values higher than 7 decrease
Trichoderma spp. growth and that the tested isolates pre-
ferred pH 5–7. The findings were consistent with our previ-
ous study demonstrating that Trichoderma species on
oyster mushroom (Pleurotus sp.) grew better under acidic-
to-neutral conditions.[28] Generally, neon light had an
inhibitory effect on the tested isolate growth, which agrees
with other reported data.[29]
According to criteria of Gea et al.,[30,31] the tested
Trichoderma isolates from Serbia and Bosnia-Herzegovina
were sensitive to chlorothalonil, carbendazim and ipro-
dione (except two isolates) since their ED50 values were
less than 5.0 mg L¡1, weakly resistant to thiophanate-
methyl (except one isolate) with ED50 higher than 5.0 mg
L¡1, while as many as seven isolates were resistant to tri-
floxystrobin, its ED50 exceeding 50.0 mg L¡1. The isolate
T. harzianum T54 was the most sensitive to chlorothalonil,
T. artroviride T60 to carbendazim and Trichoderma sp.
group 3 T90 to iprodione. Toxicity of thiophanate-methyl
and trifloxystrobin was inferior to them. T. artroviride
T33, T. aggresivum f. europaeum T85, T. harzianum T52
Trichoderma sp. group 1 T1, Trichoderma sp. group 2 T63,
and Trichoderma sp. group 3 T71 and T90 were resistant
to trifloxystrobin.
Previous ED50 quantification of fungicide toxicity to A.
bisporus F56 had enabled determination of a selective tox-
icity beteween the pathogen and A. bisporus.[18,22] In our
previous study, prochloraz manganese had demonstrated
high toxicity to all Trichoderma isolates as its ED50 values
were below 1 mg L¡1.[2] The medial ED50 value of pro-
chloraz manganese for the Serbian Trichoderma isolates
was 0.16 mg L¡1, its toxicity to A. bisporus F56 was
2.97 mg L¡1, and the selective toxicity to the pathogen
and the host 0.05.[2,18] Carbendazim toxicity to A. bisporus
F56 was 16.58 mg L¡1 and it demonstrated the best selec-
tive toxicity of 0.02 among the tested fungicides.[18] Chlor-
othalonil showed a moderate selective toxicity of 0.16,
having ED50 to A. bisporus F56 2.39 mg L¡1. Iprodione
and thiophanate-methyl, having the respective toxicity to
A. bisporus F56 of 2.14 and 12.49 mg L¡1, had the worst
selective toxicity of 1.24.[22] Trifloxystrobin toxicity to A.
bisporus was not tested because Trichoderma isolates
were weakly sensitive or resistant to that fungicide. The
 612
commercial A. bisporus strains cultivated across Europe
since the 1990s seem to be more tolerant to fungicides in
vitro than earlier ones.[32]
Chlorothalonil, iprodione and carbendazim have also
been highly effective against Serbian Cladobotryum den-
droides, while Lecanicillium fungicola var. fungicola iso-
lates have shown weak resistance to iprodione.[18,20,22]
However, thiophanate-methyl was characterized by low
activity against Serbian C. dendroides isolates, and triflox-
ystrobin was not toxic at all.[18,22]
Trifloxystrobin belongs to strobilurins, the site-specific
compounds carrying a high resistance risk. Their mode of
action, based on inhibition of mitochondrial respiration, is
assumed to be easily overcome by fungi as in the studies
concerning Trichoderma and Cladobotryum.[19] It have
been found that carbendazim and prochloraz were effec-
tive against T. harzianum from Croatia.[15] Carbendazim
toxicity against Trichoderma from Pakistan has been con-
firmed in in vitro studies.[33] Earlier studies have also
Downloaded by [FU Berlin] at 15:43 24 June 2015
demonstrated that spawn treatment by benomyl and car-
bendazim, alone or in combination with prochloraz,
reduced substrate colonization by T. aggressivum f. aggres-
sivum, in North America.[34–38] Over time, the pathogen
has developed resistance to benomyl and tiophanate-
methyl.[17] Benomyl and carbendazim have a limited appli-
cability because they are susceptible to microbial degrada-
tion, which results in reduced pathogen control.[39,40] Two
chlorothalonil formulations have been found to signifi-
cantly inhibit the growth of T. harzianum in Poland.[41]
Likewise, some rarely used fungicides (ferulic acid, men-
thol and thymol) have been reported as having fungistatic
effects on T. harzianum isolates from Croatian A. bisporus
and P. ostreatus farms, but they inhibited the host mush-
rooms as well.[15] Prochloraz was found to inhibit the
growth of aggressive Trichoderma isolates without having
a significant negative effect on the cultivated mush-
rooms.[2,15] Also, prochloraz manganese showed higher
selective fungitoxicity (0.01) to Cladobotryum dendroides
and A. bisporus than all other fungicides applied to mush-
rooms.[18] The fungicide prochloraz manganese maintains
a balance between the benefit of disease control and poten-
tial for reducing the mushroom yield.
Extended use of fungicides leads to development of
pathogen resistance and there is a need for new fungicides
to be used in practice, while preference is given to biofungi-
cides. Thus, the biofungicide based on Bacillus subtilis, has
been shown to have highly toxic effect on Trichoderma
species from Serbia, including T. aggressivum, in contrast
to that based on tea tree oil, which did not exhibit signifi-
cant antifungal capacity.[2] Their effectiveness was also
demonstrated in mushroom growing rooms, but it was sig-
nificantly lower than that of prochloraz manganese.[2]
Commercial fungicides are still being used in mushroom
farms and research studies have contributed by defining
and recommending the most efficient fungicides and their
Kosanovi

c et al.
optimum concentrations for control of green mould dis-
ease, and excluding the least effective ones.
Conclusions
After in vitro testing of five fungicides: iprodione, chloro-
thalonil, carbendazim, trifloxystrobin, and thiophanate-
methyl, carbendazim was shown to be the most effective
against Serbian Trichoderma isolates. Carbendazim was
the least toxic fungicide to A. bisporus and, besides pro-
chloraz manganese, had the best selectivity to the patho-
gen and the host. The strobilurin fungicide trifloxystrobin
was ineffective as a growth inhibitor of the tested Tricho-
derma isolates. As previous studies had reported, the fungi-
cide prochloraz manganese has the highest antifungal
effect on the causal agents of green mould. The Tricho-
derma isolates tested in this study preferred acidic-to-neu-
tral conditions, pH 5–7, and growth in the dark.
Funding
This study was funded by the Ministry of Education, Sci-
ence and Technological Development of the Republic of
Serbia, projects TR 31043 and ON 173032.
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