Impact of Fungicides Used for Wheat Treatment on Button Mushroom Cultivation

Ivana Potočnik1, Jelena Vukojević2, Mirjana Stajić2, Dejana Kosanović1,2, Emil Rekanović1, Miloš Stepanović1 and Svetlana Milijašević-Marčić1 1Institute of Pesticides and Environmental Protection, Banatska 31B, 11080 Belgrade, Serbia (ivana.potocnik@pesting.org.rs) 2University of Belgrade, Faculty of Biology, Institute of Botany, Takovska 43, 11000 Belgrade, Serbia Received: December 12, 2011 Accepted: March 3, 2012


INTRodUCTIoN
White button mushroom, Agaricus bisporus (Lange) Imbach, is the most commonly cultivated mushroom species in Serbia.Most of the substrates used for mushroom production are organic wastes or by-products, involving agricultural, municipal or industrial wastes.The substrate for its cultivation is normally fermented compost, mostly based on animal manure and cereal straw.The process of composting has two distinct phases.During phase I, various microorganisms break down the straw.In phase II, pests are eliminated and the substrate decomposes under the activity of thermophilic microorganisms (Chang and Miles, 2004).Mushroom industry is part of complex agricultural system: animal husbandry -wheat -edible mushroom -spent mushroom substrate (Manjunath and Korikanthimath, 2009).New EU regulations considered that organic mushrooms must be grown on organic straw and manure.At a present, transition period is being introduced to allow producers to adopt the new requirements as straw and manure cannot currently be obtained in sufficient quantities for organic production (Anonymous, 1998(Anonymous, , 2010)).
Potential chemical risks of several fungicides commonly used in wheat production in Serbia were evaluated for A. bisporus, regarding fungicide medium effective concentration (EC 50 ) (Potočnik 2006(Potočnik , 2009;;Potočnik et al., 2009aPotočnik et al., , 2009b)), in order to determine whether fungicide residues in straw could be directly or indirectly responsible for changes in mushroom yields.In addition, fungicides with different mode of action are currently used commercially in mushroom industry to provide control of many serious fungal diseases (Potočnik, 2006(Potočnik, , 2009;;Potočnik et al., 2005Potočnik et al., , 2008Potočnik et al., , 2010aPotočnik et al., , 2010b;;Tanović et al., 2009).The maximum tolerated levels of fungicide residues in grains, wheat straw and cultivated mushrooms are shown in table 1.The EC 50 values of eight fungicides: mancozeb, carbendazim, thiophanate-methyl, carbendazim + cyproconazole, carbendazim + flusilazole, captan, chlorothalonil, and trifloxystrobin, for A. bisporus are presented in table 2.  Potočnik et al. (2009a) reported that strain F56 of A. bisporus was able to grow at mancozeb, thiophanate methyl, and carbendazim concentrations of 12.50 mg l -1 , while growth was severely inhibited at concentrations of 25.00 mg l -1 and higher.The respective EC 50 values of mancozeb, thiophanate methyl, and carbendazim were 6.97, 10.04, and 16.58 mg l -1 .Growth of the edible mushroom mycelia was good at trifloxystrobin concentration of 25.00 mg l -1 and severely inhibited at 50 mg l -1 of this fungicide.EC 50 value of trifloxystrobin was 20.69 mg l -1 .Both captan and chlorothalonil at the concentration of 1.00 mg l -1 enabled mycelial growth of A. bisporus, inhibiting it severely at 10.00 mg l -1 .The respective EC 50 values of captan and chlorothalonil were 2.03 and 2.39 mg l -1 (Potočnik et al., 2009b).The cyproconazole + carbendazim concentration of 0.19 mg l -1 failed to affect A. bisporus growth, while concentrations of 0.37 mg l -1 and higher severely inhibited isolate growth.The EC 50 value of cyproconazole + carbendazim was 0.23 mg l -1 .Isolate of A. bisporus was capable to grow at flusilazole+carbendazim concentration of 0.05 mg l -1 , but growth was inhibited at 0.10 mg l -1 and higher concentrations.The flusilazole + carbendazim EC 50 for A. bisporus was 0.04 mg l -1 (Potočnik et al., 2009a).
Strain F56 of A. bisporus demonstrated the lowest sensitivity to trifloxystrobin and slightly higher to carbendazim (EC 50 values were 20.69 mg l -1 and 16.58 mg l -1 , respectively) (Potočnik et al., 2009a(Potočnik et al., , 2009b)).Diamantopoulou et al. (2006) reported that, in their study, mycelial growth of A. bisporus 2810 (Le Lion) was not affected by trifloxystrobin at concentration of 1.00 mg l -1 , while A. bisporus X22 was sensitive to the fungicide, having an EC 50 value of 1.10 mg l -1 (Chrysayi- Tokousbalides et al., 2007).Likewise, according to Chalaux et al. (1993), carbendazim was the only fungicide with a toxic effect on A. bisporus (Chalaux et al., 1993), while Chrysayi-Tokousbalides et al. (2007) reported its very low toxicity to A. bisporus X22 (EC 50 value of 23.20 mg l -1 ).However, carbendazim in a mixture with flusilazole or cyproconazole showed the highest in vitro toxicity to A. bisporus F56, with EC 50 values of 0.04 and 0.23 mg l -1 , respectively (Potočnik et al., 2009a).In the previous studies flusiazole was not found to significantly limit the growth of A. bisporus (Chalaux et al., 1993).
Fungicides chlorothalonil and captan showed higher toxicity than thiophanate-methyl, mancozeb, carbendazim and trifloxystrobin to A. bisporus F56, reported by Potočnik et al. (2009aPotočnik et al. ( , 2009b)).In other studies, the use of mancozeb, a fungicide from the group of dithiocarbamates, was not found to cause any damage to A. bisporus at any stage of cultivation (Yoder et al., 1950;Newman and Savidge, 1969).Previous results had indicated that chlorothalonil was able to induce toxicity problems in mushroom mycelial growth in vitro at concentrations between 0.50 and 2.00 mg l -1 (Challen and Elliott, 1985).However, Chalaux et al. (1993) did not observe any toxicity of that fungicide to A. bisporus strains B62, B98, and U3 at concentrations below 2.00 mg l -1 .Bhatt and Singh (1992) also noted a slight inhibitory effect of captan on growth of A. bisporus, while  (1993) reported that strains B62, B98 and U3 were more sensitive to captan and mancozeb than strain F56.They assumed that the strains widely cultivated in Europe in 1990s and later were apparently more tolerant to fungicides in vitro than the older commercial strains.Likewise, a strain-dependent sensitivity to fungicides had already been reported previously by Challen and Elliot (1985).

CoNCLUSIoN
Since the residues of a majority of fungicides used in wheat cultivation remain in straw in low quantities, it is less likely for them to have significant effects on composting and mushroom cultivation and yields.However, flusilazole and its formulation were shown to induce some toxicity to mycelia in vitro.Flusilazole and, to a lower extent, cyproconazole were reported as the only fungicides demonstrating toxic effects on A. bisporus.In vitro tolerance of A. bisporus to flusilazole was lower, than maximum residue level in straw of 0.10 mg kg -1 .
Although there are no specific data on the fate of fungicides during composting, Miller (1991) reported that composting could result in decomposition of hazardous materials.Chalaux et al. (1993) also postulated that carbendazim may be partly degraded during composting.Similarly, it can be postulated that flusilazole may be partly degraded during composting, but it should be further investigated in composting experiments.
Even if the sensitivity of A. bisporus to fungicides is generally higher in vitro than in vivo, the problems in mushroom compost production caused by fungicide residues from wheat straw have to be taken into account.In vitro tolerance of flusilazole and its formulation was lower than the maximum residue level allowed in straw.Therefore, flusilazole treatment could be a limiting factor for using straw for mushroom cultivation.However, with regard to resistance development, damage to the environment and human health, as well as the increasing production costs, special attention should be focused on developing alternative biological methods to protect crops from diseases.

Table 1 .
Maximum residue levels (MRL) of fungicides in cereal grain, straw, and cultivated mushrooms.

Table 2 .
Selected fungicides and their EC 50 values for Agaricus bisporus F56.