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FEMS Microbiology Letters 237 (2004) 311–315 www.fems-microbiology.org

Hydrogen peroxide concentrations detected in Agaricus bisporus sporocarps and relation with their susceptibility to the pathogen Verticillium fungicola Jean-Michel Savoie *, Miche`le L. Largeteau Institut National de la Recherche Agronomique, Unite´ de Recherche de Mycologie et Sećurite´ des Aliments, BP81, F-33883 Villenave dÕOrnon, France Received 5 February 2004; received in revised form 26 May 2004; accepted 28 June 2004 First published online 8 July 2004

Abstract A dry bubble is an undifferentiated structure that forms in place of mushrooms when cultures of Agaricus bisporus are contaminated by Verticillium fungicola. Hydrogen peroxide concentrations were measured in lyophilised samples of bubbles and healthy sporocarps from cultures of genetically related strains of A. bisporus. The strains the more resistant to the pathogen had the higher levels of H2O2 concentration measured in the bubbles, but the differences in the healthy sporocarps were not significant. That is an indication of a higher reaction to the pathogen in the forming sporocarps of A. bisporus strains associated with their partial resistance to V. fungicola. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hydrogen peroxide(H2O2); Mushroom; Agaricus bisporus; Verticillium fungicola

1. Introduction Active oxygen species including hydrogen peroxide are important biologically active molecules. Oxidative burst through the production of active oxygen species is well-established as a plant response to fungal pathogens [1]. Otherwise different phytopathogenic fungi use oxidative processes to attack and invade plant tissue, and active oxygen species are fungal pathogenicity factors [2]. Oxygen free radicals might also be responsible for sclerotia differentiation in fungi [3]. This apparent paradox whereby active oxygen species contributes both to pathogen virulence and host resistance strengthens the specificity of the interaction that can vary with the fungi and plants and the important role of oxidative *

Corresponding author. Tel.: +33-557-12-24-86; fax: +33-557-1225-00. E-mail address: [email protected] (J.-M. Savoie).

processes in these interactions between fungal pathogens and plants. Oxidative processes with production of active oxygen species are putative factors in host–pathogen interactions when both the host and the pathogen are fungi. Verticillium fungicola (Preuss) Hassebrauk, is a major fungal pathogen of the cultivated mushroom, Agaricus bisporus (J.E. Lange) Imbach and the causal agent of the disease commonly known as dry bubble. It produces several macroscopic symptoms on its host, including necrotic lesions with brown coloured spots or streaks, stipe blow-out, and dry bubbles. A dry bubble is an undifferentiated structure containing mycelia of both A. bisporus and V. fungicola. The degree of disease and the particular symptoms are dependent on the point of development of the mushroom crop at the time of infection [4]. The relationships between V. fungicola and A. bisporus seem to be of invasive necrotrophic nature. The parasite penetrates host cell walls by the combined

0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.06.051

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J.-M. Savoie, M.L. Largeteau / FEMS Microbiology Letters 237 (2004) 311–315

effect of wall-lytic enzymes and mechanical pressure [5]. Some strains of A. bisporus have effective natural partial resistance to V. fungicola [6]. The measurement of the level of susceptibility is however difficult because it is influenced by many factors and needs to have very well controlled conditions of cultivation or to increase dramatically the number of replications of the experiment. That is not favourable to the screening of a large progeny or a large sample of wild strains to study the genetics of this trait [7]. Antibiotic production by A. bisporus has been suggested as one of the factors of resistance to V. fungicola. A. bisporus released hydrogen peroxide in liquid medium [8] and there was a correlation between hydrogen peroxide concentration measured in compost and the mycelial biomass produced by A. bisporus [9]. Verticillium fungicola isolates have been compared for their tolerance to H2O2 showing variability in their susceptibility to the presence of hydrogen peroxide in their environment [10]. By comparison with similar experiments performed on Botrytis cinerea a plant pathogen [11], V. fungicola was susceptible to lower concentrations of H2O2. Hydrogen peroxide and other reactive oxygen species could contribute to the resistance of the mushroom A. bisporus to its fungal pathogen V. fungicola. The aims of this study were to examine the putative role of hydrogen peroxide as an active oxygen species in the resistance of A. bisporus to V. fungicola and to evaluate if measurement of this compound could be a criterion for the selection of resistant A. bisporus strains. An attempt was made to correlate hydrogen peroxide concentrations measured both in dry bubbles and healthy sporocarps and the susceptibility of various A. bisporus strains to V. fungicola.

2. Materials and methods Two groups of genetically related strains were used in two different trials. In Trial 1, five A. bisporus strains were used. Three of them (A622, A627, A628) were hybrids obtained in our laboratory by crossing a homocaryon of the white commercial hybrid U1 (Sylvan, USA) with three different homocaryons of SP104, a strain derived from wild isolates. The other ones were the parents, U1 and SP104. In Trial 2, 15 strains were compared: U1, a hybrid (H) between U1 and a wild strain of A. bisporus var. burnettii, and 13 hybrids between offsprings of H and the other sexual pole of U1 as described previously [12]. The 13 hybrids were selected among 48 hybrids tested in a preliminary study (data not shown), for covering a large scale of susceptibility to V. fungicola. The strains are maintained in the Institut National de la Recherche Agronomique/Centre Technique du Champignon collection, CGAB, France.

For pathogenicity tests during cultivation and sample collection, the A. bisporus strains were grown on commercial compost (from Renaud S.A., France) spawned with mycelium grown on rye grain at the rate of 0.8% in 0.9 m2 trays [10]. Six trays were spawned with each A. bisporus strain. The incubation was performed at 24 °C, 92 % relative humidity, for 13 days before a conventional casing layer was added on the top of the compost. Nine days after casing, temperature in the cultivation room was decreased to 16 °C. A conidial suspension of V. fungicola var. fungicola was prepared using three-week-old cultures on malt-agar medium and sprayed on the top of the casing layer 11 days after casing at a concentration of 106 conidia per m2 [10]. Mushrooms were harvested for 4 weeks and the weights of healthy or affected mushrooms (dry bubbles and stipe blow-out) were recorded for each tray. Data Analyses were performed with the SystatÒ package (SPSS Inc. USA). Variance analyses (ANOVA) and FisherÕs leastsignificant-difference tests were performed after transformation of the percentages of affected mushrooms pffiffiffi (y) into x ¼ arc sin½ y . The experiment was performed two times with two different batches of compost successively in the same facilities. Samples of healthy sporocarps and dry bubbles were collected for H2O2 detection. Tissue cubes (0.12 cm3) were cut from the cap of healthy sporocarps and from dry bubbles. The cubes were lyophilized, ground and stored in tubes before being assayed for hydrogen peroxide content. For each strain, four different sporocarps and four different dry bubbles were analysed by performing three replications of measurement per sample. For preparation of crude extracts, 10 mg of lyophilised powder were mixed with 1.5 mL of 0.1 M phosphate buffer, pH 6.0, and incubated at 80 °C for 40 min. The crude extracts were obtained as the supernatant after centrifugation for 5 min at 12,000g, 4 °C and used immediately for H2O2 assays in triplicates as above. Hydrogen peroxide concentrations were determined in these crude extracts with the horseradish peroxidase catalyzed oxidation of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-dimethylaminobenzoic acid (DMAB). Assays contained 0.25 mL of crude extract, 0.25 mL of a 20 U mL1 solution of horseradish peroxidase (Sigma), and 0.625 mL of chromophore solution containing 0.6 mM MBTH and 7.5 mM DMAB in 0.05 M phosphate buffer, pH 6.0. During incubation of this mixture for 3 min at room temperature, all the hydrogen peroxide present was consumed by the peroxidase for the oxidation of DMAB and MBTH, and absorbance at 590 nm was measured [13]. Blanks without peroxidase were used as reference and controls with 2800 U of bovine liver catalase (Sigma) mL1 of crude extracts were obtained to check that H2O2 was responsible for the chromophore oxidation in the assays. Standard curves were obtained with dilutions of H2O2 from


3. Results and discussion In trial 1, A622 and U1 were the more susceptible strains; SP104 and A627 were the less ones (Table 1). A628 had a middle position. Its level of susceptibility was significantly higher than this of A622, but it was not significantly different to U1, SP104 and A627. For H2O2 concentrations in the dry bubbles, two groups of strains were observed. U1 and A622 had low concentrations with values not significantly (P < 0.05) different to those in caps. SP104, A627 and A628 had significant (P < 0.05) higher H2O2 concentrations in the dry bubbles than U1 and A622 with values significantly higher than in healthy caps. In trial 2 including a higher number of strains, a significant positive correlation between H2O2 concentra-

Table 1 Levels of disease due to Verticillium fungicola var. fungicola on five strains of Agaricus bisporus and concentrations of H2O2 measured in healthy fruit bodies and in dry bubbles A. bisporus strains

% of diseased mushroom

1

lmol H2O2 g lyophilized powder Healthy sporocarps

U1 SP104 A622 A627 A628 a

21.8 7.4 42.7 11.0 17.4

a

B C A C BC

b

3.2 4.0 3.8 3.2 3.2

Aa Ab Aa Ab Ab

Bubbles 3.2Ba 5.6Aa 3.8Ba 5.4Aa 6.0Aa

Means of six trays in two replicated experiments of culture. Within this column, values followed by different letters differ significantly at P < 0.05 according to the FisherÕs least-significant-difference test performed after transformation of the percentages of affected pffiffiffi mushrooms (y) into x ¼ arc sin½ y . b Means from four samples with three replications of measurements per sample. Within a column, values followed by different capital letters differ significantly at P < 0.05 according to the FisherÕs least-significant-difference test. Different low case letters within a line indicate that differences between healthy sporocarps and dry bubbles were significant at P < 0.05.

tions in healthy sporocarps and in bubbles was observed (Table 2). Otherwise, there was a significant negative correlation between H2O2 concentration in bubbles and the susceptibility of the A. bisporus strains to V. fungicola (Table 2). Based on the data of susceptibility to V. fungicola, 3 groups of strains were obtained using the FisherÕs least-significant-difference test: G1 with the 9 more resistant strains, G2 with 2 strains in the middle position, and G3 including the 5 more susceptible strains (Fig. 1). Consequently, the more resistant strains had the higher levels of H2O2 concentration in the bubbles, but the differences in the healthy sporocarps were not significant (Table 3). Measurement of H2O2 concentration with MBTH + DMAB after heating at 80 °C has been used previously with a fungus producing high laccase activities [14]. This high temperature was used to inhibit laccases and other phenol oxidases interfering with H2O2

Table 2 Matrix of correlations between the three parameters measured in Trial 2

lmol H2O2 g1 sporocarps

lmol H2O2 g1 sporocarps

lmol H2O2 g1 bubbles

% diseased mushrooms

1

0.58a

0.44

(0.02)b

(0.10)

1

0.63

lmol H2O2 g1 bubbles

(0.01) a b

Pearson correlation coefficient, r Bonferroni probabilities that r is different from ±1.

G1

7 µmoles H2O2 g-1 mushroom

3 to 64 lM (Sigma, 30% stabilised) in 0.05 M phosphate buffer, pH 6.0, incubated at 80 °C for 40 min as the samples during the extraction phase. By this way the loss of H2O2 due to the heat treatment for the sample extraction was taken into account. By comparison with non heated H2O2 dilutions or addition of known quantities of H2O2 before sample extraction, it was observed that these losses were always lower than 10% (data not shown). Means of H2O2 concentrations were analysed by one-way ANOVA and FisherÕs least-significant-difference test. Linear regression analysis by the least squares method was used to determine correlations between H2O2 concentrations and rates of affected mushrooms. Analyses were performed with the SystatÒ package (SPSS Inc. USA).

313

G2

G3

6 5 4 3 2 0

20 40 60 80 % of diseased mushroom

100

Fig. 1. Susceptibility of 16 parented A. bisporus strains to V. fungicola estimated by the percentage of diseased mushrooms, and hydrogen peroxide concentrations detected in lyophilized samples of healthy sporocarps (circles) and dry bubbles (squares). G1, G2 and G3 are the three groups of strains that differ significantly in the percentages of diseased mushrooms at P < 0.05 according to the FisherÕs leastsignificant-difference test.

314


Table 3 Means of H2O2 concentrations in samples of the 3 groups of strains from trial 2 established on their significant differences in the % of diseased mushroom in pathogenicity tests Groups of A. bisporus strains

% of diseased mushrooms

lmol H2O2 g1 lyophilized powder Healthy sporocarps

G1 G2 G3

28.2C 43.4B 68.3A

a

4.4 A 3.8 A 3.7 A

Bubbles 5.3A 5.3A 4.1B

a

Means of the groups. Within a column, values followed by different letters differ significantly at P < 0.05 according to the FisherÕs least-significant-difference test.

concentration measurements. In a previous study, we observed that the quantity of hydrogen peroxide measured in lyophilised samples of A. bisporus sporocarps increased with the time and temperature of incubation for obtaining the crude extracts [15]. Oxidation of hydroquinones and organic acids by peroxidases or laccases can lead to the production of semiquinone radicals and autoxidation generates O 2 and then hydrogen peroxide [16–18]. During the extraction procedure used here, oxidation of quinones or organic acids could be responsible for the production of a part of H2O2 measured in the samples [15]. All H2O2 measured in lyophilised samples of sporocarps after heat treatment was probably not H2O2 present effectively at the sampling time but H2O2 generated during heating from quinones and active oxygen species present in the fungal tissues [15]. Consequently, this measurement is a mean to estimate an overall level of effective H2O2 plus putative generators of active oxygen species present in mushroom samples. With this method of measurement, there were some significant differences between strains in a progeny tested for their H2O2 concentrations in healthy sporocarps, but there was no significant correlation with the susceptibility of the strains to V. fungicola. Under the conditions of the experiment, a strain of A. bisporus having a low level of susceptibility to V. fungicola was a strain in which the outcome of the interactions with the pathogen was less frequently the development of a dry bubble than in a strain with a higher susceptibility level. Otherwise, the observation that significant higher H2O2 concentrations were measured in the dry bubbles of the less susceptible strains than in the dry bubbles of the more susceptible strains inside a progeny suggests a higher reaction to the pathogen in the forming sporocarps of A. bisporus strains associated with their partial resistance to V. fungicola. This strengthens the concept of the impact of oxidative processes in the systems leading to the resistance of A. bisporus to V. fungicola. Oxidative processes through the production of extracellular phenoloxidases have been implicated in the offensive/defensive strategies employed by fungi during interactions

of their vegetative mycelia [19], or during browning reactions at the surface of differentiated sporocarps of A. bisporus due to attacks of bacteria (Pseudomonas tolaasii) or of V. fungicola [20]. The present study shows that oxidative processes are also implicated in the defensive strategy employed by A. bisporus during interaction with a fungal pathogen that affects its differentiation and induces the formation of malformed sporocarps. The measurement of H2O2 concentrations in healthy sporocarps with the method presented here could be used as a marker of resistance to V. fungicola to screen A. bisporus strains with highly contrasted susceptibility, but not for genetic analyses of this trait. However, higher productions of hydrogen peroxide and active oxygen species could be one of the factors of the resistance of A. bisporus to V. fungicola. Further analyses of these compounds during the first stage of the infection of resistant and susceptible strains will contribute to strengthen the present findings.

Acknowledgement We thank Christiane Codefy and Patrick Castant for mushroom cultivation and Nathalie Minvielle for laboratory work. Special thanks are finally addressed to Dieynaba Djigo and Je´roˆme Bouscaut for their valuable assistance during their training period. This work was supported in part by a grant from the Centre Technique du Champignon, France.

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