In vitro Toxicity of Fungicides of Different Mode of Action to Agaricus bisporus (Lange) Imbach

Isolates of Agaricus bisporus strains F56 and U3 were tested for sensitivity to several selected fungicides in vitro. The analysis showed that flusilasole + carbendazim and cyproconazole + carbendazim were the most toxic fungicides to A. bisporus strain F56 with respective EC50 values of 0.04 and 0.23 mg/l. The least toxic fungicides were carbendazim (EC50 = 16.58 mg/l) and trifloxystrobin (EC50 = 20.69 mg/l) to A. bisporus F56 and benomyl (EC50 = 14.99 mg/l) to A. bisporus U3.


In vitro Toxicity of Fungicides of Different Mode of Action to Agaricus bisporus (Lange) Imbach INTRODUCTION
Production of white button mushroom (Agaricus bisporus (Lange) Imbach) in Serbia is severely afflicted by fungal pathogens. Chemical control of fungal diseases requires highly selective fungicides in order to prevent infection without affecting the growth of A. bisporus. Diseases of cultivated mushrooms have been controlled so far by dithiocarbamate (Yoder et al., 1950) and methylbenzimidazole carbamate (MBC) fungicides (Fletcher and Yarham, 1976;Gea et al., 1995), except in the case of British and Irish isolates of Verticillium fungicola and Cladobotryum spp. that have been found resistant to benomyl, carbendazim and thiabendazole (Fletcher and Yarham, 1976;Gaze, 1995;McKay et al., 1998;Grogan and Gaze, 2000). The dicarboximide fungicide iprodione has been used instead, but V. fun-gicola var. fungicola isolates resistant to that fungicide have been found in Spain (Gea et al., 1996). Prochloraz, from the group of sterol biosynthesis inhibitors (DMI fungicides), was introduced in the early 1980s owing to its ability to prevent the appearance of mycopathogenic fungi in mushroom units. It is the most commonly used fungicide in mushroom industry in EU countries and Serbia (Gea et al., 1996;Potočnik et al., 2007). However, satisfactory results in control of dry bubble and cobweb diseases were no longer observed after its widespread and continuous usage in the UK and Spain Gea et al., 2005;Grogan, 2006).
The fungicides officially recommended for mushroom cultivation in EU countries are formulations of carbendazim, prochloraz and chlorothalonil (Anonymous, 2005). Fungicide efficacy trials on cultivated mushroooms are very rarely conducted by agro-chemical companies because specially designed experimental facilities are required for appropriate evaluation. Such limited trials combined with high registration requirements have led to low availability of commercial fungicides approved in mushroom cultivation (Whitehead, 2002;Stoddart et al., 2004;Anonymous, 2005).
The benzimidazole fungicides benomyl, carbendazim and thiophanate-methyl, dithiocarbamate mancozeb and imidazole prochloraz are widely used in the Serbian mushroom industry (Milenković, 1997;Potočnik et al., 2007Potočnik et al., , 2008. The aim of this study was to investigate in vitro toxicity of the fungicides commonly used in mushroom production to A. bisporus. Several fungicides that have never been used for disease control of mushrooms in Serbia were also included in sensitivity tests in order to determine their potential toxicity to the isolates investigated.
The selected volumes of fungicide stock solutions were added to molten sterile medium (50 o C) in order to make concentration series of active ingredients ranging from 0.01 to 1000.00 mg/l. Preliminary concentrations of all selected fungicides were: 0.01 0.10, 1.00, 10.00, 100.00 and 1000.00 mg/l. Based on previous results, the concentrations selected for further study were: benomyl, carbendazim, thiophanate-methyl and trifloxystrobin 3.17, 6.25, 12.50, 25.00, 50.00 mg/l; cyproconazole + carbendazim 0.19, 0.37, 0.75, 1.50 mg/l; flusilazole + carbendazim 0.01, 0.03, 0.05, 0.10 mg/l; prochloraz manganese 0.019, 0.037, 0.075, 0.150 mg/l; mancozeb and iprodione 1.56, 3.12, 6.25, 12.50, 25.00 mg/l; chlorothalonil and captan 1.00, 5.00, 10.00, 50.00 mg/l. The plates with fungicide-amended and fungicide-free PDA were inoculated with inverted mycelium agar discs (10 mm), taken from the edge of twenty day-old cultures of A. bisporus, and incubated at 20 o C. Colony diameter was measured after twenty days of cultivation. Mycelial growth on the fungicide-amended PDA was presented as a percentage of growth in the control. Fungicide concentrations inhibiting mycelial growth by 50% (EC 50 ) were determined for each isolate. The data on fungicide concentrations and relative inhibition were analysed using probit analysis, according to Finney (1971). Three replicates per treatment were used.

RESULTS AND DISCUSSION
Toxicity of the selected fungicides to strains U3 and F56 of A. bisporus is shown in Tables 2 and 3. The iso- value of cyproconazole + carbendazim for strain F56 was 0.23 mg/l. A. bisporus F56 was capable to grow at flusilazole + carbendazim concentration of 0.05 mg/l, but growth was inhibited at 0.10 mg/l and higher concentrations. The flusilazole + carbendazim EC 50 for strain F56 was 0.04 mg/l. It has been reported that treatments with mancozeb, a fungicide from the group of dithiocarbamates, have not produced any evidence of damage to mushroom at any stage of its cultivation (Yoder et al., 1950;Newman and Savidge, 1969). This is consistent with our observations of low growth inhibiting effects of mancoz-eb. Strain F56 of A. bisporus also had low sensitivity to carbendazim. However, in the past, Chalaux et al. (1993) found that carbendazim had toxic effect on A. bisporus. Chrysayi-Tokousbalides et al. (2007) reported that strain X22 of A. bisporus also had a low sensitivity to carbendazim (EC 50 =23.20 mg/l). Flusilazole and to a lesser extent cyproconazole were the only fungicides demonstrating toxic affects on A. bisporus strain F56. In previous studies, flusilazole had not been reported to limit the growth of A. bisporus mycelium significantly (Chalaux et al., 1993). Those previous results also 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 (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. It is consistent with our results showing that the tested strain F56 was less sensitive to that fungicide (EC 50 value exceeded 2 mg/l). Bhatt and Singh (1992) found that captan had a slightly inhibitory effect on the growth of A. bisporus. Chalaux et al. (1993) reported that strains B62, B98 and U3 of A. bisporus were more sensitive to captan and mancozeb than strain F56 in our study. They assumed that the strains, which were widely cultivated in Europe in the 1990s and later, were apparently more tolerant to fungicides in vitro than the older commercial strains used in previous studies. A strain-dependent sensitivity of A. bisporus to fungicides has already been reported (Challen and Elliot, 1985). Strain F56 of A. bisporus was found to have moderate susceptibility to trifloxystrobin as its EC 50 exceeded 20.00 mg/l, while strain U3 was more sensitive to this fungicide (EC 50 = 5.20 mg/l). Diamantopoulou et al. (2006) reported that mycelial growth of an A. bisporus strain 2810 (Le Lion) on casing medium in tubes was not affected by trifloxystrobin at 1.00 mg/l. Chrysayi-Tokousbalides et al. (2007) found that strain X22 of A. bisporus was more sensitive to that fungicide than the strains tested in our study, as the EC 50 of this strain was 1.10 mg/l. Even if sensitivity is generally higher in vitro than in vivo, problems with mushroom mycelia growth caused by fungicide residues in casing layer have to be taken seriously. On the other hand, regarding resistance development, damage to the environment and human health risks, as well as increasing production costs, special attention should be focused on developing alternative biological methods for control of mushroom disease.