In vitro toxicity of selected fungicides from the groups of benzimidazoles and demethylation inhibitors to Cladobotryum dendroides and Agaricus bisporus

Abstract

Twenty microfungal isolates were collected from diseased fruiting bodies of Agaricus bisporus sampled from Serbian mushroom farms during 2003–2007. Based on morphological characteristics and pathogenicity tests, the isolates were identified as Cladobotryum dendroides. The isolates of C. dendroides and A. bisporusF56 and U3 were tested for sensitivity to several selected fungicides in vitro. C. dendroides isolates were found to be more sensitive to prochloraz manganese and flusilazole + carbendazim than to the other fungicides tested (EC₅₀ values were 0.09 and 0.11 mg L^{− 1}, respectively) and weakly resistant to thiophanate-methyl (EC₅₀ values ranged between 6.53 and 12.09 mg L^{− 1}). Selectivity indexes of the tested fungicides on both C. dendroidesand A. bisporusindicated that thiophanate-methyl, cyproconazole + carbendazim and flusilazole + carbendazim had much less selective fungitoxicity than benomyl, carbendazim and prochloraz manganese.

Keywords:

• Button mushroom
• cobweb disease
• fungicides
• antifungal activity

Introduction

The soil-inhabiting cosmopolitan fungi Cladobotryum spp. are the causal agents of cobweb disease of button mushroom (Agaricus bisporus (Lange) Imbach).^{[ 1 , 2 ]} The disease symptoms are cottony fluffy white or yellowish to pink colonies on mushroom casing, rapid colonization of casing surface, covering of host basidiomata by mycelia, and their decay.^{[ 3 ]} Cobweb disease is currently one of the most serious diseases in Serbian mushroom farms affecting product quality and yield.^{[ 4 ]}

At the beginning of usage of fungicides from the group of methyl benzimidazole carbamates (MBC) in the late 1960s, excellent control of many fungal diseases was noted.^{[ 5 , 6 , 7 ]} MBC fungicides interfere with the division of cell nuclei by disrupting the assembly of tubulin into microtubules.^{[ 8 , 9 ]} After several years of their intensive usage, resistant strains of target fungi started to appear.^{[ 8 ]} The resistance of cobweb disease agents to MBC fungicides, especially to benomyl and carbendazim, was reported after their extensive usage in the Republic of Ireland in 1992.^{[ 3 , 9 ]} In 1994/95, when cobweb disease took epidemic proportions in British mushroom farms, the majority of Cladobotryum spp. isolates were strongly resistant to thiabendazole and weakly resistant to carbendazim.^{[ 6 , 10 ]}

Imidazole demethylation inhibitors (DMIs), one of which is prochloraz manganese, provide effective prevention of Verticillium fungicola, Mycogone perniciosa and Cladobotryum spp. by inhibiting the demethylation step in ergosterol biosynthesis, an essential compound responsible for the stability and functioning of lipoprotein membranes in many fungi.^{[ 11 , 12 , 13 , 14 ]} Prochloraz manganese is the most effective fungicide in prevention of Cladobotryum spp., but it has been found no longer able to control the spotting symptoms of cobweb disease.^{[ 15 ]}

Benomyl, carbendazim, thiophanate-methyl and prochloraz manganese are frequently applied in Serbian mushroom farms, while thiabendazole, which is a popular fungicide in Europe, is not registrated in Serbia.^{[ 4 ]}

Since resistance to MBC fungicides has developed and the number of available fungicides is decreasing, carbendazim, prochloraz manganese and chlorothalonil are still officially recommended for mushroom cultivation in EU countries. Likewise, as studies of fungicides efficacy on cultivated mushrooms by agrochemical companies are very rare, few fungicides are currently used commercially in the mushroom industry.^{[ 16 , 17 , 18 , 19 ]} The aims of this study were: isolation of Cladobotryum spp. in A. bisporusfarms in Serbia during 2003–2007, examination of their morphological and pathogenic characteristics, and study of their and sensitivity of A. bisporusto selected fungicides.

Materials and methods

Isolates and growth conditions

The isolates of Cladobotryumcollected from diseased A. bisporus fruiting bodies in Serbia during 2003–2007 are presented in Table 1. Isolation was done by cutting off small pieces (2 × 2 × 5 mm) of fruiting bodies with disease symptoms, immersing them in a 1% sodium hypochlorite solution for 1 min, and placing on potato dextrose agar (PDA). The isolates were kept on PDA at 5°C, in a culture collection of the Institute of Pesticides and Environmental Protection, Belgrade. Colony morphology was studied after three days of cultivation on malt extract agar (MEA) at 25°C. Conidium size, septa numbers, the presence or absence of conspicuous basal hilum, as well as chlamidospore and microsclerotium production were studied. The influence of temperature on growth was studied by isolates growing on MEA, at 20°C, 25°C, and 28°C for 3 days. Three replicates per each treatment and isolate were used for statistical analysis. The data obtained were tested for significant differences by analysis of variance (ANOVA), and mean values were separated by Duncan's honestly significant difference procedure.

Table 1 List of isolates of Cladobotryumspp. and their origins.

Isolate code	Origin of isolate	Year of collection
SP1C4	Smederevska Palanka, Serbia	2003
P3C1	Pozarevac, Serbia	2003
Ba1C1	Beograd – Banjica, Serbia	2004
B1C1	Beograd – Savski Venac, Serbia	2004
Ku1C1	Kurjace, Serbia	2004
NSl1C1	Novi Slankamen, Serbia	2004
OB1C2	Ovcar Banja, Serbia	2004
OB1C3	Ovcar Banja, Serbia	2004
P7C1	Pozarevac, Serbia	2004
Res1C1	Resnik, Serbia	2004
VG3C2	Vracev Gaj, Serbia	2004
Beč1C1	Becej, Serbia	2006
Jak1C1	Jakovo, Serbia	2006
Kal1C1	Kaludjerica, Serbia	2006
NSl2C1	Novi Slankamen, Serbia	2006
Bec2C1	Becej, Serbia	2007
Veg2C1	Veliko Gradiste, Serbia	2007
VG2C2	Vracev Gaj, Serbia	2007
NSl3C1	Novi Slankamen, Serbia	2007
NSl4C1	Novi Slankamen, Serbia	2007

Pathogenicity test

Pathogenicity assay was performed on harvested basidiomes of A. bisporus.^{[ 20 ]} Spore suspensions of approximately 1 ml containing 3 × 10⁶ conidia mL^{− 1} were prepared from four-day old cultures of all tested Cladobotryumisolates. Pilei were converted and inoculated at the sites of previously removed stipes. Sterile H₂O was used as a negative control. Inoculated pilei were incubated at room temperature (22 ± 2°C) and symptom development was monitored.

Fungicides

The most commonly used fungicides in the Serbian mushroom industry: benomyl (Benfungin WP, 500 g kg^{− 1}, Galenika-Fitofarmacija), thiophanate-methyl (tested formulation WP, 700 g kg^{− 1}, Agromarket), prochloraz manganese (Octave WP, 500 g kg^{− 1}, Bayer Crop Science) and carbendazim (Galofungin WP, 500 g kg^{− 1}, Galenika-Fitofarmacija), were tested in this study. Cyproconazole and flusilazole in their respective mixtures: cyproconazole + carbendazim (Alto Combi SC, 160 g L^{− 1} + 300 g L^{− 1}, Syngenta) and flusilazole + carbendazim (Alert-S SC, 125 g L^{− 1} + 250 g L^{− 1}, Syngenta), which have never been used in mushroom farms in Serbia, were also included in sensitivity testing in order to determine their potential toxicity to the isolates obtained.

Antifungal activity

Cladobotryumisolates and isolates of A. bisporusF56 and U3 were grown on PDA amended with the fungicides. The selected volumes of stock solution of fungicides were added to molten (50°C) sterile culture media prior to pouring, grading the concentration range of active ingredients from 0.01 to 1000.00 mg L^{− 1}.^{[ 10 ]}

Preliminary concentrations of all selected fungicides were: 0.01 0.10, 1.00, 10.00, 100.00 and 1000.00 mg L^{− 1}. Based on previous results, the concentrations selected for further study were: benomyl 0.19, 0.37, 0.75 and 1.50 mg L^{− 1}; prochloraz manganese 0.019, 0.037, 0.075 and 0.150 mg L^{− 1}; carbendazim and cyproconazole + carbendazim 0.19, 0.37, 0.75 and 1.50 mg L^{− 1}; flusilazole + carbendazim 0.01, 0.03, 0.05 and 0.10 mg L^{− 1}; thiophanate-methyl 6.25, 12.50, 25.00 and 50.00 mg L^{− 1}. Each plate was inoculated with an inverted mycelium agar disc (10 mm) taken from the edge of four day-old cultures of Cladobotryumisolates and 20 days-old cultures of A. bisporus F56 and U3, placed centrally onto the fungicide-amended and fungicide-free media and incubated at 20°C. Benomyl and prochloraz manganese toxicities were tested on A. bisporus U3, and thiophanate-methyl, carbendazim, cyproconazole + carbendazim and flusilazole + carbendazim on A. bisporusF56. Three replicates per treatment were used. Colony diameter of Cladobotryum and A. bisporusisolates was measured after three and 20 days of cultivation, respectively. Mycelial growth on the fungicide-amended media was measured as a percentage against control. Fungicide concentrations inhibiting mycelial growth by 50% (EC₅₀) were determined for each isolate and data on fungicide concentrations and relative inhibition were processed by probit analysis.^{[ 21 ]} Selectivity index for each active ingredient was calculated as a ratio of mean EC₅₀ for Cladobotryumisolates and the corresponding estimate for A. bisporus.^{[ 16 ]}

A fungicide concentration that completely inhibits mycelium growth is defined as the minimum inhibitory concentration (MIC) and it was determined by measuring mycelium growth of Cladobotryumisolates on the 7th, 14th, and 21st day of incubation. The minimum fungicidal concentration (MFC) was defined after 7 days of reisolation on fungicide-free PDA. The MIC and MFC of the tested fungicides were studied by macrodilution method on fungicides-amended PDA, where concentrations of active ingredients ranged from 1.00 to 1000.00 mg L^{− 1}.^{[ 22 ]} Three replicates per treatment and isolate were used.

Results and discussion

Symptoms

Diseased fruiting bodies of A. bisporus with symptoms resembling cobweb disease were observed in 13 Serbian mushroom farms. Infection intensity depended on the development stage of fruiting bodies. Early symptoms were noticed as round, fleshy, yellowish brown lesions on A. bisporuscaps. Late symptoms progressed when the parasitic fungus formed white cobweb circular colonies on dead or damaged pinheads, spread on the surface of the casing, and covered entirely A. bisporusfruiting bodies. The fluffy mycelia, with age, become thicker, dense and granular, taking on a pinkish hue. A. bisporuscaps turned dark brown and shrunk due to soft rot. These symptoms correspond with symptoms caused by Cladobotryumisolates.

Pathogenicity test

Pathogenicity assay on mushroom pilei showed that each of the twenty isolates had high virulence level for A. bisporus. The symptoms were not produced when sterile H₂O was used as a negative control. All isolates induced severe disease symptoms on A. bisporus pilei. The growth of the pathogenic mycelia was recorded two days after inoculation. White cobweb mycelium extended beyond the inoculation site. Three days after inoculation, the sporocarps were completely covered with white cottony mycelium with profuse sporulation, resembling the symptoms of natural infection. The pilei were completely rotten, soft and decayed on the fourth day of incubation. There were no significant differences in levels of symptom development among the different isolates.

Isolate identification

The isolates formed white, cottony, aerial mycelium on MEA at 25°C. The fungus sporulated and mycelium turned yellow three days after inoculation. After a few more days, conidia and colonies turned reddish. The hyphae were hyaline, septate and prostrate, with 3–4 pointed and oppositely placed branches. The conidiophores were erect, hyaline, simple, arising from aerial mycelium. They were branching irregularly or verticillately, terminating in groups of phialides that tapered toward the apex. Conidia were hyaline, ovoid to oblong, and had one to three septa, with central or occasionally laterally placed conspicuous basal hilum. Their dimensions were 6.15–11.07 μ m × 12.30–27.06 μ m. Chlamidospores and microsclerotia were present. Maximum mycelial growth of the studied isolates was noted on MEA at 25°C when radial growth rate was ranging between 14 and 20 mm day^{− 1}. According to morpho-physiological characteristics, all investigated isolates were identified as Cladobotryum dendroides (Bull.: Fr.) W. Gams & Hooz. Identification was consistent with previous studies.^{[ 3 , 23 , 24 , 25 ]} This is a first record in Serbia of C. dendroides, a pathogen of A. bisporus.

Antifungal activity of different fungicides

The isolates were collected from crops treated with the fungicides prochloraz manganese or benomyl, which are routinely used in Serbian mushroom farms and insignificantly toxic for A. bisporus.^{[ 4 ]} Carbendazim and thiophanate-methyl had been used for many years, but their application has become sporadic now. Cyproconazole + carbendazim and flusilazole + carbendazim, never used in practice for disease control, were included in this study in order to determine their selective fungitoxicity to both C. dendroidesand A. bisporus.

The results of the sensitivity tests of C. dendroides isolates to six selected fungicides are shown in Table 2. The data show high sensitivity of all tested isolates to benomyl. They grew well at benomyl concentration of 1.00 mg L^{− 1}, while growth of the majority of isolates was severely inhibited at its concentrations of 2.00 mg L^{− 1} and higher. The EC₅₀ values were between 0.14 and 0.97 mg L^{− 1}, while MICs and MFCs were 4.00 mg L^{− 1} for all tested isolates. Isolate growth was good at carbendazim concentration of 1.50 mg L^{− 1} and severely inhibited at concentrations of 2.00 mg L^{− 1} or higher. The EC₅₀ values were between 0.24 and 2.92 mg L^{− 1}, and the concentration of 5.00 mg L^{− 1} had both inhibitory and lethal effect on all studied isolates. The results for benomyl and carbendazim are consistent with a previous study involving Indian Cladobotryumspp. isolates.^{[ 23 ]} Contrary to our results, a majority of Cladobotryumisolates from the Republic of Ireland and the UK were found to be resistant to MBC fungicides after outbreaks of cobweb disease that reached epidemic proportions in 1994/5.^{[ 3 , 10 ]} The Irish isolates were tolerant to benomyl and carbendazim at concentrations up to 10.00 mg L^{− 1}.^{[ 3 ]} At the height of cobweb epidemic in the UK in 1995, 25% of the isolates were weakly resistant to thiabendazole (EC₅₀ values ranging between 1.00 and 10.00 mg L^{− 1}) and sensitive to carbendazim (EC₅₀ < 1.00 mg L^{− 1}), while the remaining 75% were strongly resistant to thiabendazole (EC₅₀ > 200.00 mg L^{− 1}) and weakly resistant to carbendazim (EC₅₀ values ranged between 2.00 and 10.00 mg L^{− 1}).^{[ 10 ]} Thiabendazole was not included in our study as it is not commonly used in Serbian mushroom farms.

Table 2 Effects of six fungicides on Cladobotoryum dendroides growth.

	EC50 (mg L^− 1)
Isolate	Benomyl	Thiophanate-methyl	Carbendazim	Prochloraz manganese	Cyproconazole + Carbendazim	Flusilazole + Carbendazim
SP1C4	0.32	6.87	0.24	0.08	0.33	0.02
P3C1	0.19	9.43	1.38	0.04	0.67	0.08
Ba1C1	0.33	6.53	0.93	0.04	1.27	0.10
B1C1	0.23	7.27	0.92	0.08	0.46	0.03
Ku1C1	0.32	8.87	0.82	0.05	1.42	0.06
NSl1C1	0.68	10.42	0.47	0.06	1.01	0.07
OB1C2	0.97	7.78	1.27	0.04	1.82	0.04
OB1C3	0.15	6.76	1.13	0.01	2.00	0.05
P7C1	0.37	12.09	1.14	0.02	0.95	0.05
Res1C1	0.40	7.22	0.71	0.05	1.13	0.06
VG3C2	0.57	7.96	1.89	0.06	1.03	0.05
Beč1C1	0.14	10.26	2.92	0.09	0.56	0.11
Jak1C1	0.39	7.90	1.37	0.04	0.78	0.03
Kal1C1	0.43	6.87	0.55	0.04	1.45	0.07
NSl2C1	0.77	11.09	0.07	0.02	1.13	0.08
Bec2C1	0.09	7.96	0.37	0.02	0.22	0.01
Veg2C1	0.51	6.46	2.26	0.08	0.99	0.03
VG2C2	0.31	6.52	0.34	0.02	0.81	0.04
NSl3C1	0.28	13.08	1.45	0.04	1.04	0.06
NSl4C1	0.15	7.66	1.03	0.02	0.81	0.03

The analyzed isolates showed a certain ability to tolerate thiophanate-methyl. They were able to grow at its concentration of 25.00 mg L^{− 1}, but growth was severely inhibited at 55.00 mg L^{− 1} or higher. The EC₅₀ values were between 6.53 and 12.09 mg L^{− 1} and MICs and MFCs were 300.00 mg L^{− 1}. Studies of phytopathogenic fungi have shown that resistance to MBC fungicides is associated with amino acid substitutions in several distinct regions within the β -tubulin molecule due to specific mutation points at molecular level.^{[ 26 , 27 ]} Simple amino acid substitution in any one of the three main regions of the β -tubulin gene could stop the binding of benzimidazole fungicides without any effect on β -tubulin function.^{[ 28 ]} A mutation point was detected on the β -tubulin gene in C. dendroidesisolates resistant to benzimidazole fungicides, causing amino acid substitution from tyrosine to cysteine.^{[ 3 ]} Sensitivity tests of the Serbian C. dendroides isolates have shown that although they were weakly resistant to thiophanate-methyl, cross resistance was observed neither to benomyl nor carbendazim. Cross resistance to MBC fungicides had been detected in the mushroom pathogen Verticillium fungicola, which was found highly resistant to thiabendazole and benomyl.^{[ 29 ]} The molecular structures of thiabendazole and thiophanate-methyl are different from benomyl and carbendazim, and their abilities for binding to tubulin may also be different.^{[ 30 ]}

Isolate growth was good at cyproconazole + carbendazim concentration of 1.50 mg L^{− 1} and severely inhibited at concentrations of 2.00 mg L^{− 1} or higher. The EC₅₀ values were between 0.33 and 1.82 mg L^{− 1}. Fungistatic concentration was 5.00 mg L^{− 1} and lethal 10.00 mg L^{− 1}. C. dendroides isolates were strongly susceptible to flusilazole + carbendazim and prochloraz manganese. They were able to grow well at concentrations of 1.00 and 0.15 mg L^{− 1}, respectively, but were severely inhibited at 2.00 and 0.20 mg L^{− 1}, respectively, and higher concentrations. The EC₅₀ values for flusilazole + carbendazim were between 0.02 and 0.11 mg L^{− 1}, and MICs and MFCs were 10.00 mg L^{− 1}. Prochloraz manganese concentrations between 0.02 and 0.09 mg L^{− 1}presented EC₅₀ values, while those exceeding 1000.00 mg L^{− 1}were MICs. Prochloraz manganese was still very toxic for the Serbian C. dendroides isolates. These isolates were more sensitive than 25% of the British isolates whose EC₅₀ values had been found to range from 0.14 to 7.8 mg L^{− 1}.^{[ 10 ]} Cyproconazole and flusilazole are components of two tested mixtures with carbendazim, whose EC₅₀ values were lower than 2.00 and 0.11 mg L^{− 1}, respectively. Prochloraz manganese and flusilazole + carbendazim mixtures showed the highest toxicity to C. dendroides isolates due to the DMI supplements that amended the effect of carbendazim. The tested isolates, collected from Serbian mushroom farms over a period of four years, did not show statistically significant differences in sensitivity to the same fungicide (Table 2).

The analysis of in vitro toxicity of the studied fungicides to A. bisporus F56 showed that flusilasole + carbendazim and cyproconazole + carbendazim were the most toxic fungicides with EC₅₀ values 0.04 and 0.23 mg L^{− 1}, respectively (Table 3). The least toxic fungicides were tiophanate-methyl (EC₅₀ = 10.04 mg L^{− 1}) and carbendazim (EC₅₀ = 16.58 mg L^{− 1}) to A. bisporus F56 and benomyl (EC₅₀ = 14.99 mg L^{− 1}) to A. bisporusU3. In previous studies it had been reported that flusilazole did not significantly limit the growth of A. bisporus mycelium.^{[ 31 ]} In addition, A. bisporussensitivity to fungicides depended on the strain used in a test.^{[ 31 ]} A. bisporus F56 had low sensitivity to carbendazim with EC₅₀ value of 16.58 mg L^{− 1}, and a higher sensitivity to prochloraz manganese (EC₅₀ = 2.97 mg L^{− 1}). A. bisporus X22 had low sensitivity to carbendazim (EC₅₀ = 23.20 mg L^{− 1}) and intermediate to prochloraz manganese (EC₅₀ = 5.08 mg L^{− 1}).^{[ 16 ]} The selectivity indexes indicated that thiophanate-methyl, cyproconazole + carbendazim and flusilazole + carbendazim had much less selective fungitoxicity to both C. dendroidesand A. bisporusthan benomyl, carbendazim and prochloraz manganese (Table 3). There were no significant differences in the selective indexes of benomyl, prochloraz manganese and carbendazim.

Table 3 In vitro selectivity of six fungicides for Cladobotryum dendroides and Agaricus bisporus.

	EC50 (mg L^− 1)
Fungicide	Cladobotryum dendroides	Agaricus bisporus	Selectivity index∗
Benomyl	0.38	14.99	0.02 d∗∗
Tiophanate-methyl	8.45	10.04	0.84 c
Carbendazim	1.06	16.58	0.06 d
Prochloraz manganese	0.04	2.97	0.01 d
Cyproconazole + Carbendazim	0.99	0.23	4.30 a
Flusilazole + Carbendazim	0.05	0.04	1.25 b

Conclusion

The reason for the increase in cobweb disease outbreaks is still unknown. The results showed the Serbian isolates to be sensitive to cyproconazole + carbendazim, benomyl, carbendazim, prochloraz manganese, and especially to flusilazole + carbendazim, while sensitivity to thiophanate-methyl, which is no longer widely used in Serbian mushroom industry, decreases. Benomyl showed satisfactory selective fungitoxicity to both C. dendroidesand A. bisporus, but it has been withdrawn from the market. Growers in Serbia have observed that cobweb disease appears usually on the 44th day after casing when the casing layer has been treated with benomyl, and after 60 days when carbendazim is applied, despite benomyl's higher in vitro toxicity, compared to carbendazim. Cyproconazole + carbendazim and flusilazole + carbendazim may be recommended as effective against C. dendroides in Serbia after in vivo testing. The best selectivity to both the pathogen and the host had prochloraz manganese and carbendazim. Since the consequence of continuous fungicide usage is pathogen resistance, disease risks can be minimized by maintaining a higher level of hygiene in mushroom farms. Today, the introduction of new fungicides of biological origin creates new possibilities for crop protection from fungal pathogens.

Acknowledgment

This study was carried out within projects financially supported by the Ministry of Science and Technological Development of the Republic of Serbia.

Notes

∗Selectivity indexes were derived by dividing the EC₅₀ values for Cladobotryum dendroides with the corresponding values for Agaricus bisporus;

∗∗Values of selectivity indexes followed by the same letter do not differ significantly; P = 0.05, according to the Duncan test.