Anne Marie Farnet Da Silva

Leila Qasemian

Frédérique Peter-Valence

Florence Ruaudel

Jean-Michel Savoie

Sevastianos Roussos

Isabelle Gaime-Perraud

Fabio Ziarelli

Elisée Ferré

Anne-Marie Farnet
email: anne-marie.farnet@imbe.fr

Fre ´de ´rique Peter-Valence

´e Ferre

Les conditions de stockage du blanc influencent-elles le potentiel de colonisation d'un substrat a `base de paille de ble´par Agaricus subrufescens ?

Keywords: bacteriology, mycology, parasitology, virology/Microbiologie : bacte ´riologie, mycologie, parasitologie, virologie Do Almond mushroom Lignocellulolytic enzymes Mushroom cultivation Solid-state NMR Champignon amande Enzymes lignocellulolytiques Culture

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus

subrufescens?

Introduction

Mushroom cultivation is an important biotechnological process that combines the production of nutraceuticals with organoleptic and potentially medicinal properties using a biotechnological process that recycles agricultural by-products. For all these reasons, the production of Agaricus species has been widely developed and, in European countries, the button mushroom A. bisporus is the main cultivated species.

However, an emerging Agaricus species, A. subrufescens Peck, also named A. blazei Murrill sensu Heinemann, A. rufotegulis Nauta or A. brasiliensis Wasser, M. Didukh, Amazonas & Stamets [START_REF] Kerrigan | Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms[END_REF], is actively cultivated in Brazil since the early 1990s, first in the Sao Paolo State and then extended to other regions close to the Atlantic coast. It had become an important export production for this country. This species exhibits an almond flavor probably because of benzaldehyde and benzyl alcohol [START_REF] Mata | Preservation of Agaricus subrufescens strains at low temperature by using cultures on sorghum grains[END_REF], which can be of great value as foods or food flavoring materials. Moreover, edible mushrooms are known to have bioactive compounds in their biomass, such as polysaccharides with anti-cancer or tumor suppressive activity, and extracts from the basidiocarp of this species have been shown to reduce tumor size in rats [START_REF] Stijve | Characterization of flavour and taste compounds in Agaricus blazei Murill sensu Heinem., the cultivated almond mushroom[END_REF].

Interestingly, A. subrufescens can be cultivated on composts originally made for the cultivation of the button mushroom [START_REF] Jumes | Effects of Agaricus brasiliensis mushroom in Walker-256 tumor-bearing rats[END_REF]. The inoculum used for the culture in compost is made of mycelium grown under sterile conditions on cereal grains and is known as the spawn. The type of material used for the spawn (wheat, rye or millet grains. . .) and its preparation have already been described as a crucial parameter in mushroom cultivation [START_REF] Largeteau | The medicinal Agaricus mushroom cultivated in Brazil: biology, cultivation and non-medicinal valorization[END_REF]. During this key step, the fungus fully colonizes the solid carrier chosen, starts to produce lignocellulolytic enzymes and thus fungal biomass is obtained in large amount to further inoculate the culture substrate [START_REF] Mamiro | The influence of spawn type and strain on yield, size and mushroom solids content of Agaricus bisporus produced on non-composted and spent mushroom compost[END_REF][START_REF] Royse | Effects of spawn, supplement and phase II compost additions and time of re-casing second break compost on mushroom (Agaricus bisporus) yield and biological efficiency[END_REF]. The spawn is generally produced by specific companies and sent to mushroom growers, which implies their storage for several weeks before use. Unfortunately, A. subrufescens suffers damage when it is exposed for prolonged periods at temperatures of 4 8C or lower [START_REF] Kerrigan | Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms[END_REF], showing a decrease in growth ability because of precocious ageing [START_REF] Ruiz-Rodrı ´guez | Ligninolytic enzymes activities of oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates[END_REF]. Consequently, mushroom growers have difficulties in maintaining spawn under refrigeration for long-time periods without loss of mycelium viability and spawn makers recommend to store it at temperatures around 12 8C. However, there is no information on the consequences of storage at these temperatures on the quality of the spawn and the further ability of mycelium to colonize wheatstraw-based substrates. In order to thoroughly define the process, which could be recommended for its cultivation, we investigated whether spawn of A. subrufescens produced on rye seeds stored at 10 or 15 8C for 15 or 30 days was affected in its substrate-colonizing potential of horse manure and wheat-straw-based compost. The hypothesis that sustains this work is that A. subrufescens has a threshold of tolerance for storage conditions defined via both temperature and time of storage. The substrate-colonizing potential of six strains after different storage conditions was tested using microbiological and chemical markers: both laccase and cellulase activities, which are the main extracellular enzymes responsible for the transformation of the lignocellulosic material, and the chemical characterization of the substrate using 13 C solid-state NMR spectroscopy, which give information about the dynamics of this biological transformation. Moreover, the functional diversity of bacterial communities of the compost using catabolic level physiological profile (CLPP) was investigated, since this microbial part of the substrate is essential for mushroom production under solid-state fermentation conditions. It is thus important to focus on substrate microbial communities since they are involved in both substrate transformation and selectivity. We also included in this study an A. bisporus strain commonly cultivated in France as control of substrate colonization.

Material and methods

The fungal strains

Six strains of Agaricus spp. from the Collection of germplasms of Agaricus in Bordeaux (CGAB), INRA were used: strains CA454, CA560, CA572, CA646 and CA647 for A. subrufescens and one strain Bs527 for A. bisporus. CA454 is a subculture of the collection strain WC837 in PSUMCC registered as A. blazei and assumed to be similar to ATCC 76739, which is claimed to be a copy of the original strain cultivated for the first time in Brazil in the 1960s. CA646 and CA647 are two cultivars sold in Europe by Mycelia (Belgium) under the reference numbers 7700 and 7703, respectively. CA560 was isolated in Botucatu, Brazil (1999) from a commercial farm and CA572 is a Brazilian cultivar purchased by D.C. Zied in 2007; both strains have proved to be genetically different [START_REF] Foulongne-Oriol | Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens[END_REF]. Their original reference numbers are ABL-99/28 and ABL-07/58, respectively, in the Culture Collection of the Edible and Medicinal active ´es apre `s stockage en comparaison avec le contro ˆle ainsi que la transformation du substrat, comme de ´crit par les ratios d'aromaticite ´et d'humification calcule ´s a `partir des donne ´es de RMN. Ce re ´sultat indique qu'une croissance myce ´lienne a probablement eu lieu durant la pe ´riode de stockage a `10 ou 15 8C, conduisant a `une plus grande quantite ´de biomasse dans l'inoculum. De plus, la diversite ´fonctionnelle microbienne du substrat a e ´te favorise ´e, montrant que l'e ´lectivite ´du substrat e ´tait maintenue. Ainsi, ces recherches indiquent que des recommandations aupre `s des producteurs de champignons peuvent e ˆtre formule ´es pour la culture d'Agaricus subrufescens dans les conditions standard utilise ´es en Europe.

ß 2014 Publie ´par Elsevier Masson SAS pour l'Acade ´mie des sciences.

Mushroom Module of Sa ˜o Paulo State University (UNESP). An A. bisporus strain, Bs527, for which storage is commonly performed at 2-4 8C, was used as control. Bs527 was a copy of the cultivar 30A (Euromycel) placed in the CGAB in 1997.

Mesocosm experimental set-up

Precultures were performed in glass Petri dishes containing 12 g dry weight of cooked and coated rye grains (spawn substrate used by spawn makers, provided by Somycel, Langeais, France) inoculated with 6 plugs (1 cm  1 cm) of malt agar cultures. They were incubated in a climatic chamber at 25 8C and 90% RH (relative humidity) for 15 days. Then, to evaluate the effect of two factors i.e. time (15 or 30 days), and temperature (10 or 15 C) of storage, these precultures were incubated in a climatic chamber either at 10 8C or 15 8C for 15 or 30 days and were used immediately after storage to inoculate the mesocosms. For controls, the precultures were directly used to inoculate the mesocosms. These mesocosms were prepared with 450 g of fresh mushroom compost (135 g dry weight) placed in a 2-L glass pot in order to obtain a final volume of compost of 20 cm  16 cm  6 cm (length  width  height). They were inoculated with 3% (w/w) of rye grains (dry weight), which were thoroughly mixed with the compost under aseptic conditions. The compost was commercial compost produced by SARL Renaud et Fils at Avy, France, for the cultivation of A. bisporus. The main raw materials used were horse manure and wheat-straw, and the phase I of composting was performed indoor. Compost quality was assessed by the yields obtained in commercial cultivation facilities that reached standards. Three mesocosms were prepared for each strain and were incubated in a climatic chamber at 25 8C and 90% of RH for 15 days in darkness. An uninoculated control (with the compost alone), UIC, was also prepared and incubated under the same conditions.

Lignocellulolytic activity assays

For each mesocosm after incubation at 25 8C for 15 days, enzyme extraction was performed as previously described by Velazquez et al. ( 2004) using 10 g of a composite sample (10 samplings per mesocosm) in a 1-L flask containing 200 mL of an extraction solution (polyvinylpolypyrolidone 5.7 g, CaCl 2 , 0.2 M, Tween 80, 0.05%). These samples were subjected to axial shaking for 1 h at 120 rpm at room temperature. The solids were eliminated by centrifugation at 10,000 g for 15 min and filtration through Whatman GF/D filters (2.7 mm) and through Whatman GF/C filters (1.7 mm). The filtrates of each extract were concentrated overnight at 4 8C in dialysis tubes (15 kda cut-off) using polyethyleneglycol to a final volume of 10% of initial volume.

Laccase activities were measured by monitoring the oxidation of syringaldazine to quinone (e M = 65,000 M À1 Ácm À1 ) at 525 nm in acetate buffer, 0.1 M, pH 5 on a spectrophotometer Biomate 3 (Fischer Bioblock Scientific). One unit per mL (U.mL À1 ) of laccase activity is defined as the amount of mmol of quinone of syringaldazine produced per min and per mL of sample at room temperature. Three assays were performed for each enzyme extract.

CM-cellulase activities were measured with carboxymethylcellulose (CMC) and using a simplified methodology adapted from that of Somogyi-Nelson, as previously described [START_REF] Farnet | A modified method based on arsenomolybdate complex to quantify cellulase activities: application to litters[END_REF]. Solution A was composed of 50 mL of CuSO 4 , 2%, Na 2 SO 4 , 4 g, complemented with 50 mL of a solution of Na 2 CO 3 , 25 g, NaHCO 3 , 20 g and sodium potassium tartrate, 25 g, q.s.p. 1 L. Solution B was composed of ammonium-molybdate, 25 g, H 2 SO 4 , 21 mL, and Na 2 HAsO 4 7H 2 O, 3 g, q.s.p. 450 mL. To measure CMcellulase activity, 0.1 mL of the extract was incubated at 50 8C for 1 h in 0.9 mL of 50 mM acetate buffer (pH 5.0) with 0.1% CMC. 1 mL of solution A was added to 1 mL of the reaction medium and 1 mL of distilled water; then the mixture was boiled for 20 min and cooled down in a cold water bath for 15 min. Then, 1 mL of solution B was added and the mixture was left for 50 min at room temperature and then centrifuged for 2 min at 10,000 g. The absorption was measured at 870 nm. Dilutions were performed when necessary. For the calibration curves, 1 mL of solution A was added to 1 mL, 750 mL, 500 mL, 250 mL of a solution of glucose (80 mg/L) q.s. 1 mL. Three assays were performed for each enzyme extract.

Microbial catabolic profiles via Biolog EcoPlate TM

For each mesocosm after incubation at 25 8C for 15 days, 10 g of a composite sample (10 samplings per mesocosm) were vigorously shaken in 100 mL of a sterilized desorption-solution (sodium pyrophosphate 0.08 g/L) for 1 h and then brought to a final OD = 0.001 at 595 nm. One hundred and fifty microliters of the extracted solution were used to inoculate all 96-wells of a BIOLOGß EcoPlate (Biolog, California, USA). One EcoPlate TM was used for three mesocosm extracts, since each 96-well plate contained three replicates, each with 31 different carbon sources, one per well, and a water blank. The plates were incubated at 25 8C for 48 h and the absorbance was read at 590 nm using a microplate reader (Tecan, France). To measure the functional diversity (FD), the Shannon's diversity index [START_REF] Shannon | A mathematical theory for communication[END_REF] was calculated [START_REF] Zack | Functional diversity of microbial communities: a quantitative approach[END_REF] from: FD = -S p i (ln p i ), where p i is the ratio of color development of well i to the sum of color development of all positive wells. The microbial activity of each microplate was assessed as average well color development (AWCD) calculated as follows: AWCD = S OD i /31, where OD i is the optical density for each well.

2.5. The cross-polarization magic angle spinning 13 C nuclear magnetic resonance (CP/MAS 13 C NMR) procedure An amount of 10 g of substrate, sampled as described above, were lyophilized and then ground and sieved (1 mm). CP/MAS 13 C NMR spectra were obtained on a Bruker DSX 400 MHz spectrophotometer operating at 100.7 MHz. Then, samples (600 mg) were spun at 10 KHz at the magic angle. Contact times of 2 ms were applied with a pulse width of 2.8 ms and a recycle delay of 3 s.

Chemical shift values were referenced to glycine signal (carbonyl C at 176.03 ppm). The 13 C NMR spectra were divided into seven chemical shift regions, according to [START_REF] Ziarelli | Applications et de ´veloppements des me ´thodes de re ´sonance magne ´tique nucle ´aire haute re ´solution a `l'e ´tat solide pour les mate ´riaux organiques et inorganiques[END_REF]. Dmfit 2003 software was used to determine the intensity of each chemical shift region [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF]. An index of decomposition (HR#1 Alkyl-C/O-Alkyl-C) was calculated as described before [START_REF] Baldock | Assessing the extent of decomposition of natural organic materials using solid-state C-13 NMR spectroscopy[END_REF]. An aromaticity ratio was also calculated to estimate the degree of humification of organic matter and Alkyl-C/COOH-C ratio HR#2 was used to assess organic matter transformation using the alkyl chain length of organic acids.

Statistical analysis

The significance of differences (enzyme activities, AWCD and H 0 from Biolog, NMR ratio) between the means of three experiments was tested by non-parametric multiple comparison of Kruskal-Wallis. Each experiment was considered as an independent sample. The optical absorbances of the 30 wells in the ECO plate were subjected to Principal Component Analysis (PCA) after 48 h of incubation, considering each substrate as a variable. Statistica Vs 6 (StatSoft, Maison-Alfort, France) was used for statistical analysis and a P-value < 0.05 is considered as significant.

Results and discussion

Potential of substrate colonization after spawn incubation using microbial and chemical markers

Agaricus subrufescens is known to be sensitive to low temperatures, and this particular characteristic is an issue to define adequate storage conditions [START_REF]CT Agaricus blazei cultivation for a living in Brazil[END_REF]. Thus, ideal storage conditions, with limited consequences on the ability to colonize cultivation substrate, were still to be defined. Substrate colonization can be assessed by monitoring the extracellular enzyme involved in lignocellulosic material transformation [START_REF] Farnet | Capacity for colonisation and degradation of horse manure and wheat-straw-based compost by different strains of Agaricus subrufescens during the first two weeks of cultivation[END_REF]. For these lignocellulolytic activities, the storage of the spawn had an effect on cellulase and laccase activities measured in the composted substrate: after certain storage conditions, these activities were indeed enhanced. Fig. 1 A and B shows the tendency observed for the A. subrufescens strains studied, using the results obtained for strains CA572 and CA 647 for cellulase and laccase activities, respectively. Cellulase activities were significantly higher than in controls (no storage of the spawn) when spawn was stored for 30 days at 10 or 15 8C (for instance with strain CA 572 in Fig. 1A). Concerning laccase activities, storage had no effect or led to higher laccase activities after spawn incubation at 15 8C for 30 days (Fig. 1B with strain CA 647). In non-inoculated compost, no laccase and cellulase activities were detected. Thus, surprisingly, storage at cold temperatures seems to have a positive effect on the production of the extracellular enzymes involved in the transformation of lignocellulosic substrate during spawn running. The chemical transformation of the substrate was monitored via solid-state NMR of 13 C: different ratios [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF][START_REF] Baldock | Assessing the extent of decomposition of natural organic materials using solid-state C-13 NMR spectroscopy[END_REF], an aromaticity ratio (AR) and two humification ratios (HR1 = Alkyl-C/O-Alkyl-C and HR2 = Alkyl-C/COOH-C) were calculated from the NMR spectra to determine how storage conditions of the spawn may affect the capacity of transformation of the substrate. For AR, when an effect was observed, it was always a higher ratio in the case of substrate colonized by spawn previously stored at 10 or 15 8C than in control (Table 1). The same tendency was observed for HR1. Higher values in both AR and HR1 indicate that the recalcitrant fraction of organic matter (linked to alkyl-C and aromatic-C signals and thus assigned to lipids and phenolic compounds) accumulated in the substrate, while O-Alkyl-C signal, assigned to polysaccharides, decreased. These results clearly indicate that the easily biodegradable fraction of organic matter, i.e. mainly polysaccharides, is more effectively transformed in the substrate inoculated with spawn previously stored at 10 or 15 8C. Thus, our results obtained with solid-state NMR of 13 C corroborated those found with lignocellulolytic activities, both microbial and chemical markers indicating that organic matter is actively transformed when spawn is previously stored. For HR2, a higher ratio in substrate inoculated with pre-incubated spawn than in control shows that long alkyl chain acids were predominantly observed. These molecules can be linked to phospholipids from cell membranes [START_REF] Peter-Valence | Chemical characterization of the biomass of edible medicinal mushroom Agaricus subrufescens, via solid-state 13 C NMR[END_REF] and this suggests that fungal biomass may be more extensively produced in solid-state fermentation under these conditions, i.e. when the inoculum has been previously stored at 10 or 15 8C for two or four weeks. Spawn storage at 10 or 15 8C seems to enhance the potential for colonization of the culture substrate during spawn running, as described by both microbial and chemical markers used here. This can also be linked with the efficient polysaccharide transformation shown by the increase in both the AR and HR1 ratios.

A. subrufescens grows at higher optimal temperatures than A. bisporus: for instance in previous studies [START_REF] Colauto | Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation[END_REF], the authors found that optimal temperatures for mycelial growth was around 30 8C, depending on the strains studied. De Mendoca et al. [START_REF]CT Agaricus blazei cultivation for a living in Brazil[END_REF] reported optimal temperatures ranging from 25 to 28 8C and Llarena et al. [START_REF] Llarena-Hernandez | Potential of European wild strains of Agaricus subrufescens for productivity and quality on wheat-straw based compost[END_REF] optimal temperatures varying from 26 to 30 8C, with a similar range of variation for cultivars and wild strains. Long-time storage at low temperatures such as 4 8C is known to be lethal for A. subrufescens [START_REF] Zou | Effect of Zn supplementation on the growth, amino acid composition, polysaccharide yields and anti-tumour activity of Agaricus brasiliensis[END_REF]. Mycelial growth rate decreased gradually over storage time at 4 8C of spawn on sorghum grains used for long-term storage of genetic resources [START_REF] Mata | Preservation of Agaricus subrufescens strains at low temperature by using cultures on sorghum grains[END_REF]. Here, we found that storage at 10 or 15 8C for a period as long as 30 days did not strongly affect the potential of different A. subrufescens strains in their ability of early colonization and transformation of the compost used as cultivation substrate during the spawn running period.

Changes in functional diversity of microbial communities from the substrate inoculated with spawn previously stored at 10 or 15 8C

To thoroughly investigate the consequences of mycelium storage on A. subrufescens ability to colonize compost and how it may change its biotic environment, the diversity of microbial communities of the substrate was also considered via their functional structure. Ecoplates are known to focus on the functional diversity of the bacterial communities since the reduction of tetrazolium dye cannot be accomplished by fungi [START_REF] Heuer | Evaluation of community-level catabolic profiling using BIOLOG GN microplates to study microbial community changes in potato phyllosphere[END_REF][START_REF] Preston-Mafham | Analysis of microbial community functional diversity using sole-carbon-source utilization profiles: a critique[END_REF]. Thus, the Agaricus strain, which colonizes the culture substrate, is not supposed to be involved in the catabolic potential measured here. Global microbial activity was measured by Average Well Color Development (AWCD) and functional diversity via the Shannon-Weaver Index, H 0 [START_REF] Shannon | A mathematical theory for communication[END_REF][START_REF] Zack | Functional diversity of microbial communities: a quantitative approach[END_REF]. As for lignocellulolytic activities, a similar tendency was observed for all the A. subrufescens under study and Fig. 2A and B shows the results obtained with strains CA 454 and 647 as an example. In most of the treatments, AWCD was the same as in control. When AWCD varied from the control, higher values were observed whatever the temperature of storage considered (Fig. 2A). For functional diversity characterized by H 0 , the same result was observed: a higher H 0 value was found when spawns were stored at 10 8C or 15 8C than in control (Fig. 2B). Thus, storage of the spawn had probably an effect on A. subrufescens mycelium, which consequently influenced the microbial communities during mycelial colonization of compost, by enhancing both their global catabolic activity -and thus their potential to transform the substrate -and their biodiversity. Moreover, the high diversity of microbial communities of the substrate is of major importance in the selectivity of the mushroom culture substrate, since it prevents the colonization of potential antagonists [START_REF] Vela ´zquez-Ceden ˜o | Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates[END_REF]. Fig. 3 shows the result of PCA analysis using optical densities of all the substrates used as variables. Axes F1 and F2 accounted for 59.39 and 10.42% of the variance, respectively. This figure shows that the temperature used for storage (10 or 15 8C) did not globally affect microbial catabolic diversity, since the projections were not clearly differentiated on the PCA (squares vs triangles). On the other hand, the time of storage (15 or 30 days) influenced this diversity (black vs white) mainly according to axis F2, as observed for strains CA647 and CA560, and to a lesser extent according to F1, for strains CA572 and CA646. PCA was used to give additional information about how the functional diversity of these communities varied. Though the effects of both time and temperature of storage were observed, the more drastic conditions, i.e. 10 8C and 30 days, did not lead to a strong variation in the diversity structure of substrate microbial communities according to the PCA. Colonization of the substrate by the mycelium of A. subrufescens may have modified the associated microbial communities because of microbial interactions, including competition for nutrients or production of antagonistic metabolites. Here, we found that the diversity index, H 0 , remained constant or was even higher after spawn storage and PCA added complementary information by indicating that for certain strain, this functional diversity actually changed compared to control.

A. subrufescens can be considered as an important edible mushroom because of its medicinal and culinary properties, which culture conditions still have to be precisely described. In our previous study [START_REF] Farnet | Capacity for colonisation and degradation of horse manure and wheat-straw-based compost by different strains of Agaricus subrufescens during the first two weeks of cultivation[END_REF], we found that a horse manure and wheat-straw-based compost produced under physicochemical conditions commonly used for the button mushroom A. bisporus was suitable for this tropical Agaricus species. Here, our investigations focused on storage conditions of the spawn, which is a very important parameter to favor mushroom cultivation. Our study revealed that storage conditions of the spawn for up to 30 days, at relatively low temperatures for this fungus, did not further affect substrate colonization by this species. Storage under certain conditions even improved its potential of colonization and this potential seems to be strain dependent. These results are encouraging, since they demonstrate that these storage conditions at 10 to 15 8C cannot be ruled out in the cultivation process. However, further studies should determine whether these storage conditions have effects on crop yields or mushroom quality in order to confirm these first findings.

  A.-M.Farnet et al. / C. R. Biologies xxx (2014) xxx-xxx 3 G Model CRASS3-3280; No. of Pages 8 Please cite this article in press as: A.-M. Farnet, et al., Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?, C. R. Biologies (2014), http://dx.doi.org/10.1016/ j.crvi.2014.06.002

Fig. 1 .

 1 Fig. 1. (Color online.) Trends in cellulase (A) and laccase (B) activities measured in a horse manure-and straw-based compost (here, strains CA 647 and CA 572 as an example). The spawn was previously stored at 15 8C for 15 days, 15 8C for 30 days, 10 8C for 15 days, or 10 8C for 30 days or not stored (C) and then used for inoculation (incubation was then performed at 25 8C for 15 days). The letters define the mean values, which are statistically different from each other according to the multiple comparison of Kruskal-Wallis (Dunn procedure).

Fig. 2 .

 2 Fig. 2. (Color online.) Trends in (A) AWCD (Average Well Color Development) and (B) Shannon-Weaver index, H 0 , based on Ecoplate data after 48 h of incubation of the microbial extract from a horse manure, and straw-based compost (here strains CA 454 and CA 647 as an example). The spawn was previously stored at 15 8C for 15 days, 15 8C for 30 days, 10 8C for 15 days or 10 8C for 30 days or not. The letters define the mean values, which are statistically different from each other according to the multiple comparison of Kruskal-Wallis (Dunn procedure).

Fig. 3 .

 3 Fig. 3. (Color online.) PCA (based on Pearson's correlation) performed using OD at 48 h of each Ecoplate carbon source (n = 30) as variables. The two first factors (F1 + F2) accounted for 72% of total variance. UIS: Un-Inoculated substrate, horse manure-and straw-based compost inoculated with Agaricus bisporus (strain Bs527) or five strains of A. subrufescens, and incubated at 25 8C for 15 days. The spawn was previously stored at 15 8C for 15 days (C1, ), 158 C for 30 days (C2, ), 10 8C for 15 days (C3, ) or 10 8C for 30 days (C4 ) or not (C, ).

  A.-M.Farnet et al. / C. R. Biologies xxx (2014) xxx-xxx 7 G Model CRASS3-3280; No. of Pages 8 Please cite this article in press as: A.-M. Farnet, et al., Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?, C. R. Biologies (2014), http://dx.doi.org/10.1016/ j.crvi.2014.06.002

Table 1

 1 Aromaticity ratio (AR) and two humification ratios, HR#1, Alkyl-C/O-Alkyl-C and HR#2, Alkyl-C/COOH-C, calculated from the relative intensities of chemical functions from 13 C NMR data from horse manure-and straw-based substrate inoculated with five different Agaricsus subrufescens strains and A. bisporus, and incubated at 258 C for 15 days. The spawn was previously inoculated at 158 C for 15 days (C1), 158 C for 30 days (C2), 108 C for 15 days (C3) or 108 C for 30 days (C4) or not (C). The different letters indicate when values differ significantly according to the Kruskal-Wallis test and Dunn's procedure for multiple comparisons of means (P < 0.05). Please cite this article in press as: A.-M. Farnet, et al., Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?, C. R. Biologies (2014), http://dx.doi.org/10.1016/ j.crvi.2014.06.002

																			CRASS3-3280; No. of Pages 8	G Model
		Strains
		Bs 527			CA 454			CA 560			CA 572			CA 646			CA 647
	AR
	C	0.204	AE 0.011	ab	0.166	AE 0.013	ab	0.196	AE 0.019	b	0.155	AE 0.007	a	0.170	AE 0.017	a	0.162	AE 0.007	a
	C1	0.200	AE 0.005	ab	0.158	AE 0.004	a	0.200	AE 0.013	b	0.191	AE 0.020	b	0.189	AE 0.014	a	0.195	AE 0.004	b
	C2	0.196	AE 0.005	a	0.162	AE 0.007	ab	0.183	AE 0.012	ab	0.189	AE 0.005	b	0.186	AE 0.005	a	0.179	AE 0.011	ab
	C3	0.200	AE 0.006	ab	0.202	AE 0.007	b	0.175	AE 0.006	a	0.176	AE 0.006	ab	0.182	AE 0.012	a	0.159	AE 0.033	a
	C4	0.208	AE 0.004	b	0.178	AE 0.029	ab	0.178	AE 0.008	ab	0.185	AE 0.010	b	0.180	AE 0.014	a	0.187	AE 0.005	ab
	HR1
	C	0.413	AE 0.017	c	0.209	AE 0.011		0.407	AE 0.060	a	0.302	AE 0.011	a	0.370	AE 0.012	a	0.361	AE 0.062	a
	C1	0.365	AE 0.009	ab	0.185	AE 0.011		0.344	AE 0.035	a	0.382	AE 0.011	bc	0.348	AE 0.043	a	0.380	AE 0.017	a
	C2	0.336	AE 0.037	a	0.224	AE 0.046		0.349	AE 0.024	a	0.375	AE 0.018	abc	0.407	AE 0.075	a	0.372	AE 0.036	a
	C3	0.374	AE 0.020	abc	0.352	AE 0.044		0.357	AE 0.032	a	0.323	AE 0.028	ab	0.399	AE 0.026	a	0.314	AE 0.058	a
	C4	0.404	AE 0.017	bc	0.331	AE 0.052		0.337	AE 0.009	a	0.404	AE 0.034	c	0.355	AE 0.064	a	0.395	AE 0.064	a
	HR2
	C	2.856	AE 0.056	ab	1.121	AE 0.043	a	2.122	AE 0.109	a	2.051	AE 0.207	a	2.220	AE 0.222	a	1.930	AE 0.136	a
	C1	2.819	AE 0.278	ab	1.720	AE 0.297	ab	2.450	AE 0.116	b	2.409	AE 0.153	b	2.454	AE 0.144	a	2.602	AE 0.022	b
	C2	2.716	AE 0.105	a	1.928	AE 0.125	ab	2.263	AE 0.046	ab	2.420	AE 0.231	b	2.359	AE 0.243	a	2.592	AE 0.197	b
	C3	2.911	AE 0.122	ab	2.695	AE 0.054	b	2.427	AE 0.292	ab	2.082	AE 0.080	ab	2.113	AE 0.311	a	2.437	AE 0.381	ab
	C4	2.973	AE 0.118	b	2.541	AE 0.360	b	2.125	AE 0.132	a	2.178	AE 0.209	ab	2.199	AE 0.359	a	2.414	AE 0.137	ab

A.-M. Farnet et al. / C. R. Biologies xxx (2014) xxx-xxx 5

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (French Government founds), project ANR-09-BLAN-0391. We are very grateful to Mrs Marjorie Sweetko for her very helpful assistance in English language.


Dr Stéphane Gastaldi
email: stephane.gastaldi@univ-amu.fr

Ibrahima Cissokho

Anne-Marie Farnet

Elisée Ferré

Michèle P Bertrand

Gérard Gil

Enzyme-triggered Radical Reactions: Another Approach For Tin-free Radical Chemistry

Keywords: 1,3-Dicarbonyl compounds, Laccase, Oxidoreductase, Radical mediator, Radical reaction

The system laccase/mediator/dioxygen is able to trigger radical reactions with radical precursors which are not natural substrates of this enzyme. The radical generation has been accomplished by single electron transfer oxidation of a 1,3-dicarbonyl precursor. The process is exemplified with a radical cascade.

Over the last few years, our research group has explored the possibility to broaden the synthetic potential of radical reactions by combining them with an enzymatic transformation. This led us to report the first switchable dynamic kinetic resolution (DKR) process allowing the synthesis of either (R)-or (S)-amides from racemic amines depending on the nature of the enzyme, lipase or protease. [1] This fruitful adventure in the world of hydrolases led us to think about the possibility to use an enzyme to initiate radical reactions. The latter can be initiated through a redox reaction. [START_REF] Kita | Radical in Organic Synthesis[END_REF] This second approach might be achieved with the assistance of an oxidoreductase. Indeed, numerous redox reaction, such as oxidation of alcohol, aldehyde or ketone, hydroxylation of aromatic or nonactivated carbon atoms, can be catalyzed by isolated enzymes. [START_REF] Monti | [END_REF][START_REF] Hollmann | For a review on enzyme-mediated oxidation[END_REF] Oxidase, a subclass of the oxidoreductases, is constituted of any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen as the final electron acceptor. [5] In these reactions, oxygen is reduced to water or to hydrogen peroxide. In order to avoid the formation of hydrogen peroxide in the reaction medium that might induce side reactions, laccases have been selected for this preliminary study since, with these copper-containing oxidases, [5b,6] the only side-product formed during the redox process is water (Scheme 1).

Laccases have a low redox potential (0.5-0.8 V/NHE) [6,7] which explains why their natural substrates are mainly electron-rich phenols. [6a] To extend the scope of applications to non-phenolic substrates, a redox mediator can be used (Fig. 1). The role of mediators in laccase oxidations is depicted in Scheme 2. [8] They act as 'electron shuttles'; after being oxidized by the enzyme, the oxidized form of the mediator oxidizes any substrate that could not enter in the enzymatic pocket.

Moreover, the wide structural variety of oxidized mediators enables the reaction to evolve through oxidation mechanisms that cannot be catalyzed by the enzyme alone, and thus to broaden the range of potential substrates (Scheme 2). Thus PT and ABTS radical cations lead to single electron transfer (SET) reactions (Fig. 1). [9] N-Hydroxy mediator-derived radicals (e.g. HBT, HPI) enable the abstraction of a hydrogen atom from benzylic positions. [9a,10] In the present paper, we describe our recent results on the use of the laccase-mediator system to initiate a radical reaction on radical precursors which are not laccase classical substrates.

SET oxidative processes that enable the generation of radicals are interesting moiety (6). No transformation occurred in the presence of EPT contrary to the reactivity previously observed with unsubstituted 1,3-dicarbonyl compounds.

On the other hand, when a solution of 6 in toluene was added to the dark blue buffered solution of ABTS •+ , a slow discoloration of the solution was observed. ABTS was added by portion until the blue color became persistent (0.6 equiv.). After consumption of the substrate (TLC monitoring, 3 h), the reaction was stopped and analyzed. Cyclopropane 7 was characterized as a mixture of two diasteromers (1:1). Its formation might be explained by the mechanism proposed in Scheme 5. The α-dicarbonyl radical C, resulting from the oxidation of 6, added reversibly to the double bond. Equilibrium was then displaced by the oxidation of radical D with ABTS •+ leading to the benzylic cation E which was trapped by a molecule of water to form 7 in 38% yield. This radical-polar crossover cascade showed that ABTS was able to act first as 'electron shuttle' between the laccase and the radical precursor and second as final oxidant. However the modest yield and the quantity of mediator used in the process, 0.6 equiv., confirmed previous observations that ABTS might give rise to byproducts and therefore it was not the best mediator for a selective reaction. [24] In summary, this preliminary study showed that the couple laccase/mediator, with dioxygen as co-oxidant, is a viable alternative to initiate a radical reaction involving a redox process. This new method of radical generation enables the formation, at 30 °C, of C-C bond and subsequent functionalization. This system might be improved by the design of new mediators which will not interfere with the radical cascade. These results will be reported in due course.

Experimental Section

Solvents are commercially available, they were used as purchased, without further purification. Purifications were per-alternatives to tin-based methodologies. [START_REF] Dalko | For a review on SET reactions[END_REF] Carbon-centered radicals have been generated from neutral carbonyl derivatives, carbanions, enamines or enolates by employing, among other oxidants, Mn(OAc) 3 , [12] ceric ammonium nitrate (CAN), [13] copper(ii) [14] or iron(iii) [14a,b,15] complexes. The main drawback of these methodologies is that the oxidant is often used in excess and it is difficult to get rid of metal traces. This problem may be easily overcome by the enzymatic approach. Indeed, laccases enable the use of a catalytic amount of enzymes since the co-oxidant of the reaction is dioxygen. Moreover, the copper atoms in charge of the oxidation are strongly liganded to the enzyme and from this point of view the medium can be considered as metal-free at the end of the process.

The goal of this preliminary study was to trigger, with a laccase, a radical reaction from a substrate which is not a classical enzyme substrate. To reach this goal, it was necessary to select a mediator capable to act as an 'electron shuttle', between the enzyme and the substrate, during the reaction. Thus ABTS and Meldrum's acid were selected as mediator and radical precursor respectively. The strong acidity of this 1,3-dicarbonyl compound enables the use of the corresponding sodium salt, as substrate in the buffer aqueous medium required to maintain the enzyme active. The commercially available laccase Polyporus pinsitus 504 (P.p. 504) [START_REF] Yaver | The E°redox potential of P. p. 504 has been measured[END_REF] from Novo Nordisk was selected so that the methodology could easily be reproduced or extended to other substrates. When Meldrum's acid (1) or its sodium salt (Na-1) was reacted with the radical cation of ABTS (ABTS •+ ) [START_REF]E°= 0.69 V for ABTS •+ /ABTS versus NHE[END_REF] (1 equiv.), generated in the presence of P. p. 504 in a pH 5 succinate buffer solution, no evolution, like dimerization, was detected and the dark blue color of ABTS •+ remained unchanged. Another mediator, N-ethylphenothiazine (EPT), [START_REF] Ebdrup | [END_REF][START_REF] Madej | EPT potential redox was not found in the literature, but E°for similar structures are reported: E°= 1.03V for N-methylphenothiazine (MPT)[END_REF] was used under the same conditions. The light orange color of EPT •+ became violet instantaneously after addition of Na-1 before turning again light orange at the end of the reaction (24 h). The analysis of the mixture showed that ylide 2 [START_REF] Huang | Close structures have recently been reported[END_REF] together with sulfoxide 3 were formed in 59 and 16% yield respectively (Scheme 3). The same result was observed starting from 1, showing that at pH 5 the equilibrium between both forms enabled the reaction to proceed.

The formation of 2 might be explained on the grounds of the mechanism proposed in Scheme 4. After a first electron transfer (ET) step from EPT to the enzyme, EPT •+ oxidized Na-1/1 through a second ET generating radical A. The latter was then trapped by the persistent EPT •+ (B) [START_REF]For examples of coupling of an alkyl radical with a sulfur-centered radical cation[END_REF] which evolved to 2 after deprotonation. The concomitant formation of 3 resulted from the reaction of EPT •+ with water. The structure of 3 was confirmed by comparison with an authentic sample produced by a direct oxidation of EPT with m-CPBA, [START_REF] Tosa | [END_REF] in this case the corresponding sulfone was also produced. The latter was not detected during the enzyme-catalyzed reaction.

This transformation was applied to other acidic 1,3-dicarbonyl compounds. Acetylacetone and dimedone led to 4 and 5 in 58 and 78% yield (Fig. 2) whereas a less acidic substrate like ethyl cyanoacetate did not undergo any transformation. It must be underlined that the formation of sulfoxide 3 could not be avoided.

This first approach showed that a commercially available laccase could be used as catalyst to generate a carbon-centered radical. However, the nature of the couple laccase/mediator had to be optimized in order to preclude any interference of the mediator with the fate of the radical species. In spite of the rather disappointing preliminary results obtained with ABTS, we decided to reinvestigate its reactivity. The absence of evolution, when the reaction was performed with 1, did not necessarily mean that the oxidation of the dicarbonyl compound did not occur. The interesting point was that if the oxidation proceeded the resulting radical did not react with the mediator.

With this purpose in mind, Meldrum's acid was functionalized with an efficient internal radical acceptor, [23] i. each time the color turned yellow ABTS was added by portion (7 x 22 mg) until the color remained blue. Once the reaction finished (TLC monitoring, 3 h), the diphasic solution was extracted with AcOEt (3x) and dried over MgSO 4 . After concentration, the crude product was purified by flash chromatography on silica gel (10/90 to 20/80 AcOEt/pentane) leading to 7 (84 mg, 0.3 mmol, 38%) as a mixture of diasteromers, further purification enables the isolation of pure samples of each isomers for characterization. Isomer 7a: 1 H NMR (CDCl 3 ): 7.32-7.42 (m, 5H), 4.84 (d, J = 9.6, 1H), 2.60 (q, J = 9.2, 1H), 2.13 (m, 2H), 1.98 (s, 3H), 1.78 (s, 3H). 13 formed on Macherey Nagel silica gel 60 A (70-230 mesh). Analytical thin layer chromatography was performed on pre-coated silica gel plates. Visualization was accomplished by UV (254 nm) and with phosphomolybdic acid in ethanol. 1 H NMR, 13 C NMR spectra were recorded at 400 MHz and 100 MHz, respectively. Chemical shifts (δ) are reported in ppm. Signals due to residual undeuteriated solvent ( 1 H NMR) or to the solvent ( 13 C NMR) served as the internal standards: CDCl 3 (7.26 ppm and 77.0 ppm). Multiplicity is indicated by one or more of the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). The lists of coupling constants (J) correspond to the order of multiplicity assignment and are reported in Hertz (Hz). APT was used for 13 C spectra assignment.

Enzyme Assay

The crude laccase from Polyporus pinsitus (P.p. 504) from Novo Nordisk (170 mg) was diluted in 2 mL of pH 5 succinate buffer (0.2 M) and corresponds to P. p. 504 enzymatic extract in the following. Enzyme activity (1.93 μm/min/mg) was measured by monitoring the oxidation of syringaldazine [25] to the corresponding quinone (ε M = 65 000 M -1 cm -1 ) at 525 nm in succinate buffer 0.1 M, pH 4.0 on a spectrophotometer Uvikon XL. One unit (U) of laccase activity is defined as the amount of enzyme that oxidizes 1 µmol of the substrate per minute per g.

General Procedure for Ylide Synthesis

To 10-ethylphenothiazine [START_REF] Ebdrup | [END_REF] (91.2 mg, 0.4 mmol) in a mixture of acetone (3.2 mL) and pH 5 succinate buffer (32 mL) at 30 °C was added P. p. 504 enzymatic extract (100 µl). The solution was stirred 30 min before addition of 1. 3-dicarbonyl compound (0.4 mmol). After 24 h stirring, the reaction medium was extracted with CH 2 Cl 2 (3x), the combined organic phases were dried over MgSO 4 . After concentration, the crude product was purified by flash chromatography on silica gel (5/95 to 100/0 AcOEt/ pentane) leading to the title compound.

5-(10-Ethyl

-10H-phenothiazin-5- ylidene)-2,2-dimethyl-1,3-dioxane- 4,6-dione (2)
1 H NMR (CDCl 3 ): 7.60 (dd, J = 6. 4 2-(10-Ethyl-10H-phenothiazin-5ylidene)-5,5-dimethylcyclohexane-1,3-dione ( 5)

1 H NMR (CDCl 3 ): 7.44 (m superimposed, 4H), 7.17 (d, J = 8.9, 2H), 7.04 (t, J = 7.3, 2H), 4.29 (q, J = 7.1, 2H), 2.26 (s, 4H), 1.55 (t, J = 7.1, 3H), 0,96 (s, 6H). 13

Cyclization of 6

To ABTS (22.1 mg, 0,04 mmol) in pH 5 succinate buffer (32 mL) at 30 °C were added P. p. 504 enzymatic extract (400 µL), the color of the solution becomes dark blue. Then (E)-2,2-dimethyl-5-(3'phenylallyl)-1,3-dioxane-4-6-dione [23] (209 mg, 0.8 mmol) in toluene (20 mL) was added. The solution was stirred and

  Fig. 1. Examples of mediators.

  Scheme 3.

  C NMR (CDCl 3 ): 168.1 (C ester ), 166.5 (C ester ), 142.1 (C arom. ), 129.0 (CH arom. ), 128.6 (CH arom. ), 126.0 (CH arom. ), 105.9 (O-C-O), 71.7 (-CH-O), 45.5 (-CH-C), 29.4 (-C-), 27.8 (CH 3 ), 27.7 (CH 3 ), 25.5 (CH 2 ). Isomer 7b: 1 H NMR (CDCl 3 ): 7.35 (m, 5H), 4.85 (d, J = 8.4, 1H), 2.72 (q, J = 8.4, 2H), 2.13 (m, 2H), 1.98 (s, 3H), 1.78 (s, 3H). 13 C NMR (CDCl 3 ): 167.8 (-C=O ester ), 166.5 (C=O ester ), 141.8 (-C= arom. ), 129.1 (=CH arom. ), 128.9 (=CH arom. ), 126.7 (CH arom. ), 105.3 (O-C-O), 71.6 (-CH-O), 44.7 (-CH-C), 28.5 (-C-), 28.0 (-CH 2 -), 27.5 (CH 3 ), 26.5 (CH 3 ). HRMS ([M+NH 4 ] + , ESI): m/z calcd for C 15 H 20 NO 5 : 294.1336. Found: 294.1341.

  C NMR (CDCl 3 ): 191.1 (C=O), 142.5 (C arom ), 132.4 (CH arom ), 130.0 (CH arom ), 121.8 (CH arom ), 114.7 (CH arom ), 107.9 (C arom ), 99.5 (C=S), 43.5 (CH 2 -N), 31.0 (CH 2 ), 29.0 (C), 28.2 (2xCH 3 ), 12.6 (CH 3 ). HRMS ([M+H] + , ESI): m/z calcd for C 22 H 24 NO 2 S: 366.1522. Found: 366.1521.

  Scheme 5.

  CH arom ), 110.3 (C arom ), 97.8 (C=S), 43.2 (CH 2 -N), 29.7 (2xCH 3 ), 12.6 (CH 3 ). HRMS ([M+H] + , ESI): m/z calcd for C 19 H 20 NO 2 S: 326.1209. Found: 326.1208.

	and 1.6, 2H), 7.50 (ddd, J = 8.7, 7.2 and 1.6, 2H), 7.22 (d, J = 8.3, 2H), 7.11 (broad t, J = 8.0, 2H), 4.26 (q, J = 7.2, 2H), 1.53 (s, 6H), 1.56 (t, J = 7.2, 3H). 13 C NMR (CDCl 3 ): 163.0 (C=O ester ), 142.9 (C arom ), 133.2 (CH arom ), 130.7 (CH arom ), 122.4 (CH arom ), 115.4 (CH arom ), 106.8 (C arom ), 103.2 (O-C-O), 60.4 (C=S), 40.0 (CH 2 -N), 26.1 (2xCH 3 ), 12.9 (CH 3 ). HRMS ([M+Na] + , ESI): m/z calcd for C 20 H 19 NO 4 SNa: 392.0927. Found: 392.0921.
	3-(10-Ethyl-10H-phenothiazin-5-ylidene)pentane-2,4-dione (4) 1 H NMR (CDCl 3 ): 7.43 (td, J = 7.2 and 1.4, 2H), 7.38 (dd J = 7.8 and 1.2, 2H), 7.13 (m superimposed, 2H), 7.04 (t, J = 7.3, 2H), 4.22 (q, J = 7.1, 2H), 2.47 (s, 6H), 1.52 (t, J = 7.1, 3H). 13 C NMR (CDCl 3 ): 189.7 (C=O), 142.0 (C arom ), 132.0 (CH arom ), 128.5 (CH arom ), 121.8 (CH arom ), 114.8 (

Acknowledgements

Gift of laccase from Polyporus pinsitus (P.p. 504) from Novo Nordisk is gratefully acknowledged.

Received: February 21, 2012


Anne Marie Farnet Da Silva

M Roux

J Le Petit

A M Farnet

J Le

R C N R S 6116

Genotypic variations among isolates of Marasmius quercophilus, a white-rot fungus isolated from evergreen oak litter

Keywords: Marasmius quercophilus, intraspecific variability, ITS, RAPD Marasmius quercophilus, variabilité intraspécifique, ITS, RAPD

published or not. The documents may come

Genotypic variations among isolates of Marasmius quercophilus, a white-rot fungus isolated from evergreen oak litter

Introduction

Marasmius quercophilus is a white-rot fungus which colonizes evergreen oak litter, a typical Mediterranean ecosystem [START_REF] Barbero | Les chênaies vertes du sud-est de la France mediterranéenne: valeurs phytosociologiques, dynamiques et potentielles[END_REF]. This fungus has not yet been the subject of investigations, although its enzymatic properties are of great interest. [START_REF] Tagger | Phenoloxidases of the white-rot fungus Marasmius quercophilus isolated from an evergreen oak litter (Quercus ilex, L.)[END_REF] have recently described the enzymatic properties of an isolate from the site of la Gardiole de Rians (Var, France), which is widely populated by evergreen oak, and thus the involvement of this fungus in litter degradation phenomena. Indeed, they have showed the amylolytic, pectinolytic, and cellulolytic activities of this basidiomycete and also its extensive production of polyphenoloxidases such as laccases. Furthermore, the enzymatic systems involved in ligninolysis such as polyphenoloxidases have provoked considerable interest since they find applications in biotechnology (kraft pulp bleaching or aromatic pollutant degradation). In our study, 26 isolates were collected in the same site (La Gardiole de Rians). This site has been the subject of our research in many fields such as biological activity in the litter [START_REF] Chabert | Dégradation biologique de la litière de chêne vert dans deux forêts de la région provençale (Var, France)[END_REF], chemical analysis of tanins [START_REF] Racon | Histological and chemical changes in tannic compounds of evergreen oak leaf litter[END_REF], and bacterial biodiversity [START_REF] Périssol | Succession of bacteria attached to evergreen oak leaf surfaces[END_REF][START_REF] Brunel | Occurrence of Bacillus species on evergreen oak leaves[END_REF].

The objective of this work was to study a potential genotypic polymorphism in M. quercophilus species since the genetics of this fungus has not yet been investigated. Genetic variations are mainly linked to mutations, genetic exchanges, spore spread, and adaptation to the environment. Thus, this study constituted a first attempt to investigate the biology and ecology of M. quercophilus, which seems to have an important role in the evergreen oak ecosystem. Furthermore, the genetic variability among the isolates studied may explain potential differences observed in their enzymatic properties. Molecular biology provides efficient tools such as RFLP (restriction fragment length polymorphism) after am-plification of the ITS (internal transcribed spacer) fragments from the rRNA repeat, or random amplified polymorphic DNA (RAPD) [START_REF] Williams | DNA polymorphisms amplified by arbitrary primers are useful as genetics markers[END_REF]) to determine subspecies variability. The RAPD technique is not a timeconsuming and expensive method. This may explain the rapid growth of interest for this technology in behavioural ecology and population biology in the past few years. It is based on the use of random oligonucleotides with priming sites in an inverted orientation, which are close enough to permit PCR (polymerase chain reaction) amplification. Both of these methods have been applied successfully for the detection of polymorphisms and the classifi-cation for various fungal species [START_REF] Demeke | Potential taxonomic use random amplified polymorphic DNA (RAPD): a case study in Brassica[END_REF][START_REF] Fegan | Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae[END_REF][START_REF] Kumeda | Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi[END_REF][START_REF] Overmeyer | Genetic variability among isolates and sexual offspring of the plant pathogenic fungus Calonectria morganii on the basis of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)[END_REF]. Here, we report on the successful use of RAPD in the analysis of genetic diversity of the M. quercophilus species.

Materials and methods

Isolation of Marasmius quercophilus isolates

Carpophores of M. quercophilus were collected at the site of La Gardiole de Rians (Var, France), in diverse areas and on different substrata: white or evergreen oak leaves, acorns, or wood (Table 1). In this site, the four areas (about 20 m 2 ) were all 100 m distant. Dikaryotic isolates were obtained by using the rhizomorphic form of the fungus: cultures from fungal cap were obtained on a maltagar medium, 20 g•L -1 (Bio Mérieux, Marcy l'Etoile, France) containing 50 mg•L -1 chloramphenicol (Sigma). Then, pure mycelial cultures were grown on a special agar medium (whole wheat flour, 20 g•L -1 ) that favours rhizomorph production. Rhizomorphs were cultured on a malt-agar medium (20 g•L -1 ).

Enzymatic tests

Cultures were grown on mineral medium [START_REF] Kirk | Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium[END_REF]) and 0.5 g•L -1 cellulose. These cultures were homogenized with a sterile magnetic bar and were used to test for API Zym strips (Bio Mérieux, Marcy l'Etoile, France), which were incubated at 28°C for 24 h. This technique is a semiquantitative micromethod designed for the research of enzymatic activities. It allows a rapid study of 19 various enzymatic reactions. Assays for aromatic compound transformation were realized in Monod tubes as described by [START_REF] Tagger | Phenoloxidases of the white-rot fungus Marasmius quercophilus isolated from an evergreen oak litter (Quercus ilex, L.)[END_REF]. The aromatic compounds tested included p-coumaric acid, ferulic acid, and tannic acid (Sigma).

DNA extraction

Still cultures were grown in a 250-mL Erlenmeyer flask containing 50 mL of malt extract (20 g•L -1 ) at 30°C for 15 days. Cultures were filtered on microglass filters (Whatman, F/D, 2.7 µm), and the mycelia were frozen in liquid nitrogen and ground to a fine powder in a mortar. An Eppendorf tube was filled with about 0.1 g of the powdered mycelium, 0.5 mL of extraction buffer (0.5 M NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) was added, and the solution was well mixed and left to stand 10 min. The same volume of phenol and 0.3 mL of chloroform -isoamyl alcohol (2:1, v/v) were added. The mixture was shaken vigorously and left for 10 min. The suspension was centrifuged at 10 000 × g for 15 min, and the upper phase was removed carefully and transfered to a new Eppendorf tube. Ten microlitres of a 20% (w/v) solution of RNase A (Sigma) was added and the mixture was incubated for 10 min at 37°C. A volume of 0.4 mL of chloroform -isoamyl alcohol was added, and the solution was mixed and centrifuged at 10 000 × g for 10 min. The upper phase was transferred to a fresh Eppendorf tube, and one volume of isopropanol was added. The suspension was mixed by inversion and put at -20°C for 1 h. After centrifugation for 30 min, the DNA pellet was washed with 70% ethanol and centrifuged briefly. The supernatant was removed, and the pellet was dried under vacuum (10 min). The pellet was resuspended in 20 µL of TE buffer (0.01 M Tris-HCl, pH 8.0, 0.1 mM EDTA).

Amplification and sequencing of fragments ITS 1 and 2

Primers M1, M2, M3, and M4 were described by [START_REF] White | PCR protocols: a guide to methods and applications[END_REF]. Primers M1 (5′-TCCGTAGGTGAACCTGCGG-3′) and M2 (5′-GCTGCGTTCTTCATCGATGC-3′) were used for fragment ITS 1 amplification and primers M3 (5′-GCATCGATGAAGAACGCA GC-3′) and M4 (5′-TCCTCCGCTTATTGATATGC-3′) to amplify fragment ITS 2. The amplification conditions consisted of 75 mM Tris-HCl (pH 9), 20 mM (NH 4 ) 2 SO 4 , 0.01% Tween 20, 2.5 mM MgCl 2 , 5 mM of each dNTP (Eurogentec, Seraing, Belgium), 100 pmol of each primer (synthesized by Eurogentec) using either primer M1 or M2, M3 or M4 with the appropriate primer, 5 ng genomic DNA, and 1 unit of Taq Polymerase (Goldstar, Eurogentec) in a final reaction volume of 100 µL. The reaction mixtures were prepared on ice and sealed with a drop of mineral oil before thermal cycling. The amplification program was the same for the two primer pairs: 30 cycles consisting of 30 s denaturation (93°C), 60 s primer annealing (55°C), and 45 s extension (72°C). This amplification was performed using the PTC 100 Thermal Controller (MJ Research, U.S.A.). The amplicons were separated on a 1.5% (w/v) agarose gel in TAE buffer with 0.1 µg•mL -1 of ethidium bromide for 3 h at 4 V•cm -1 . The size marker was a 100-bp ladder (GenSura Laboratories, Inc.) Note: All the isolates were collected on evergreen oak leaves except for those specified. For isolate labelling, the number represented the carpophore, and the letter referred to the rhizomorph. When only one rhizomorph was isolated from the fungal cap culture, the isolate name included only a number.

Table 1. Origin of the 26 Marasmius quercophilus isolates from the site of la Gardiole de Rians (Var, France).

Each fragment was sequenced in both directions using the appropriate primer with the T7 DNA polymerase sequencing kit (Pharmacia).

RAPD conditions

The reaction concentrations were identical to those described above. Among 20 primers tested (Operon Technologies Inc., Alameda, Calif., U.S.A.) the primer OPA 09 was chosen since every isolate showed a response. The OPA 09 sequence is 5′-GGGTAACGCC-3′. The reaction mixture was amplified through 30 cycles consisting of the following optimized program: 30 s denaturation (94°C), 30 s primer annealing (33°C), and 60 s extension (72°C). We used the optional step "increment" of the PTC 100 thermocycler to realize a progressive increase in temperature (0.5°C•s -1 ) from annealing to extension. The amplified fragments were separated on a 1.5% agarose gel as described above. The band sizes were determined with the Image analysis software (Kodak Digital Science).

All amplification reactions were conducted at least three times in three separate experiments for each isolate.

Statistical analysis

Each of the RAPD bands was scored as discrete character in a binary matrix: 0 for absence and 1 for presence of reproducible where N xy is the number of fragments shared by two isolates x and y, and N x and N y are the number of fragments for isolate x and y, respectively. Hierarchical cluster analysis was based on the neighbour-joining method [START_REF] Saitou | The neighbour-joining method: a new method for reconstructing phylogenetic trees[END_REF].

Results

The use of primer pairs M1-M2 and M3-M4 led to the amplification of a single type of fragment in each case: about 320 bp for the ITS 1 fragment and about 450 bp for the ITS 2 fragment. Although the four M. quercophilus isolates sequenced did not show any differences in ITS 1 and ITS 2 sizes, the sequencing established nucleotide variability (Fig. 1): 1.25% for ITS 1 and 1.11% for ITS 2 (four nucleotides were different for fragment ITS 1, five nucleotides for fragment ITS 2). We tested the 20 primers of kit A provided by Operon Technologies, and we obtained a RAPD profile for the 26 isolates tested with oligonucleotide OPA 09. With the other primers, a maximum of four isolates reacted to the amplification process. Each profile was reproducible from one amplification to another using newly extracted DNA from new cultures. The RAPD patterns obtained with primer OPA 09 showed 24 bands whose sizes varied from 80 to 6200 bp. Using statistical analysis by Nei and Li coefficient and the neighbour joining method as hierarchical clustering, we obtained a tree (Fig. 2). We observed that the branch lengths, which are proportional to genetic distance, were quite similar. This would suggest that the M. quercophilus isolates studied were fairly close genetically. Nevertheless, numerous deep branches in the tree demonstrated the presence of a polymorphism in this population. Indeed, the tree allowed us to group closely related isolates: cluster 1, which included isolates 13, 25, 28, 2b, 2c, and 4b; cluster 2 with isolates 10, 14, and 26; cluster 3 with isolates 22 and 24; cluster 4 with isolates 1a, 6, 8, 18, 19, 21, and 27; cluster 5 with isolates 9, 17, and 23. Four other isolates were separated from the rest of the population, and they were grouped in two other clus-ters: cluster 6 with isolates 15 and 3b and cluster 7 with isolates 5a and 5b. The minimum similarity coefficient of [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF] observed in these different clusters was 0.278. The coefficient between isolates belonging to different clusters was greater, for example, the coefficient between isolates 10 and 1a was equal to 0.856 (when two isolates are different, this coefficient is equal to 1).When two isolates are identical the coefficient of [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF] is equal to 0: this was the case for some of the isolates studied, which were not differentiated (isolates 6, 18, and 19, and isolates 10 and 14). The tree also showed that isolates isolated from the same carpophore (carpophore 1, 2, 3, 4, or 5) were grouped in the same cluster. Some isolates collected from certain areas were grouped in the same clusters: clusters 1 and 4 in areas II and III and clusters 2, 5, 6, and 7 in areas I and IV.

Enzymatic tests such as acid phosphatase and β-galactosidase detection showed similar responses for all the isolates (data not shown). However, for other enzymes, we observed the same pattern in the different clusters described by the RAPD experiment. This was the case for the presence of ferulic acid (FA), tannic acid (TA), p-coumaric acid (p-CA), Fig. 2. The tree of 26 Marasmius quercophilus isolates obtained from the RAPD results using the [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF] similarity coefficient and the neighbour-joining method [START_REF] Saitou | The neighbour-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The tree was rooted using isolate 5a. The seven clusters observed are specified at the left of the tree.

esculin (E), and salicillin (S) transformation, esterase-lipase (EL), and N-acetyl β-glucosaminidase (NAG) (Table 2).

Discussion

ITS analysis did not distinguish between isolates; all the isolates were quite similar, and the sequences of fragments ITS 1 and 2 showed few differences for the four isolates tested (1.25 and 1.11%, respectively). Although both the ITS region and the intergenic spacer of the nuclear rRNA repeat unit evolve quickly and thus are useful in describing intraspecific genetic variations [START_REF] White | PCR protocols: a guide to methods and applications[END_REF], intraspecies homology has already been reported using analysis of ITS fragments. For example, in the case of both Fusarium oxysporum Schlechtendahl isolates [START_REF] Edel | Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species[END_REF] and Aspergillus [START_REF] Kumeda | Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi[END_REF] section Flavi (A. flavus Eries, A. parasiticus Speare, A. oryzae Ahlburg, A. sojae Sakagushi & Yamada, A. tamarii Kita), no differences were detected in the restriction patterns of fragments ITS 1 and 2 although isolates were collected from several different countries in both cases. Furthermore, [START_REF] Zimand | Use of the RAPD procedure for the identification of Trichoderma isolates[END_REF] have observed great homology among Trichoderma harzianum Rifai isolates which were isolated from various sites (United States, Norway, Germany, Sweden). Extensive genetic stability has also been found in the plant pathogenic fungus Calonectria morganii Crous, Alfenas & Wingfield [START_REF] Overmeyer | Genetic variability among isolates and sexual offspring of the plant pathogenic fungus Calonectria morganii on the basis of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)[END_REF] and among Metarhizium anisopliae Metschnikoff isolates [START_REF] Bidochka | Differentiation of species and isolates of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD)[END_REF].

In this study, the RAPD data showed an overall genetic stability, since the branch lengths of the tree, which are proportional to genetic distance, were quite similar. This can be explained by the intraspecific nature of the study. Extensive genetic exchanges within a population lead to a decrease in genetic diversity, since this genetic flow occurs easily between isolates of the same species. But we also observed a greater degree of homology between some isolates forming subgroups, which demonstrated polymorphism in the population studied (the minimum coefficient of similarity was 0.278). This extensive polymorphism may be due to different environmental pressures, which vary with the area where the fungi were collected. Furthermore, a progressive separation of fungal hyphae even in a quite limited site may lead to genetic divergence. These interpretations are consistent with the fact that the isolates (except cluster 3) grouped in the same cluster were collected in the same areas (II and III; I and IV). These observations suggest that the environmental influences (temperature, moisture, pH) are the same in areas I and IV and in areas II and III. Thus, the isolates grouped in clusters 1 and 4 and clusters 2, 5, 6, and 7 seem to have adapted to the same environmental factors in areas II and III and in areas I and IV, respectively. However, this hypothesis has to be considered with caution, since the environmental factors of the different areas are not yet clearly defined, and some of the isolates collected on various substrata (acorn, evergreen oak or white oak leaves) were grouped in the same cluster. [START_REF] Assigbetse | Differentiation of Fusarium oxysporum f. sp. races on cotton by random amplified polymorphic DNA (RAPD) analysis[END_REF] have also attributed the differentiation of Fusarium oxysporum f.sp. vasinfectum to the geographical location of the isolates. De la Bastide et al. (1994) have successfully distinguished Laccaria bicolor Maire isolates depending on the area where the isolates were collected. [START_REF] Rohel | variation correlated with RAPD-PCR pattern polymorphisms in the entomopathogenic fungus Erynia neoaphidis and some closely related species[END_REF] have shown that some Erynia neoaphidis Remaudière & Hennebert isolates from different continents showed extensive homology, while other isolates of the same species presented differences even when they were collected in the same area in France. This demonstrated the wide variety of results possible.

The heterogeneity of this population was illustrated by the enzymatic data: isolates belonging to the same cluster had certain similar phenotypic properties. Phenotypic potential alone can not be used to estimate the variability of a population, but it can reflect heterogeneity previously detected by genome analyses. [START_REF] Wildman | Influence of habitat on the physiological and metabolic diversity of fungi[END_REF] has also shown that the geographic location and the substrate of origin of Fusarium acuminatum Ellis & Everhart isolates influenced the morphological aspects of this fungus. Furthermore, toxin production was affected by the habitat of the different isolates of this fungal species. The Fusarium compactum Wollenweber isolates, which were also analysed in this study, differed in their carbon utilization patterns depending on the sites where they were collected.

In our RAPD study, several isolates from the same carpophore were grouped. It was expected that these isolates would be genetically close, since their genetic difference was due to meiosis alone, these dikaryons having been obtained from the recombination of the monokaryons produced by the same polysporic culture. The grouping of these isolates has thus showed the ability of the RAPD method to analyse genotype relationships at different taxonomic levels. The isolates that were considered as identical (6, 18, and 19; 10 and 14) were collected at the same time (1996). Isolates 6, 18, and 19 were collected in the same area (area II) and isolates 10 and 14 were collected in area I. The hypothesis of a clone dispersed in a site has already been described [START_REF] Turner | Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species[END_REF]. [START_REF] Cooke | Spatial distribution and temporal persistence of discrete genotypes of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton[END_REF] have also reported clone spread in ecosystems in the case of different basidiomycete populations. The somatic incompatibility loci are numerous and multiallelic. Both these situations minimize the chance of two isolates in a population having exactly the same somatic incompatibility alleles. This limits genetic exchanges and encourages the development of clonal populations.

This first attempt to analyse the M. quercophilus genome showed genetic polymorphism in this species. Further studies will help us to better describe this species, for example, using isolates from other sites or other geological substrata (such as siliceous soils). At the same time, this would allow us to investigate the potential influence of environmental factors on a fungal population.

  bands. Genetic similarity was estimated from this binary matrix with the RAPDistance program version 1.04[START_REF] Armstrong | RAPDistance programs, version 1[END_REF]) using Nei's genetic distance coefficient[START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF]

Fig. 1 .

 1 Fig. 1. The fragment ITS 1 and ITS 2 sequences of the four isolates specified: fragment ITS 1 (single strand) amplified with primer M1, fragment ITS 2 (single strand) amplified with primer M3. Different nucleotides are in boldface and underlined. The name of the isolate analysed is specified at the beginning of the sequence.

Author to whom all correspondence should be addressed. e-mail: afarnet@hotmail.com

© 1999 NRC Canada Farnet et al.

© 1999 NRC Canada

 Can. J. Bot. Vol. 77, 1999Cluster Isolate E S FA p-CA TA EL NAG Area

Note: Enzymatic tests were esculin (E), salicillin (S), ferulic acid (FA), p-coumaric acid ( p-CA), and tannic acid (TA) transformation and esterase-lipase (EL) and N-acetyl β-glucosaminidase (NAG) presence.


Anne-Marie Farnet
email: amfarnet@netscape.net

Stéven Criquet

Mireille Cigna

Gérard Gil

Elisée Ferré

Purification of a laccase from Marasmius quercophilus induced with ferulic acid: reactivity towards natural and xenobiotic aromatic compounds

Keywords: Aromatic compounds, Laccase, Mediators, White-rot fungi

In this study, we describe the biochemical features and the ability of a new laccase isoform from a Marasmius quercophilus strain collected on evergreen oak litter to transform various aromatic compounds. This laccase, induced with ferulic acid, exhibits interesting enzymatic properties, such as great thermal stability at 30 and 40 • C (24 h), no inhibition with EDTA, Cystein or SDS. These biochemical features are quite different from those observed with the laccase of another M. quercophilus strain collected in a different area of the South of France. Thus, this study shows the degree of variation in the properties of this enzymatic system within the same species. Furthermore we demonstrate that several natural aromatic compounds were transform leading to polymerization. The transformation of chlorophenols such as 2-chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol is also observed without adding mediators (2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate) or 1-hydroxybenzotriazole) to the reaction mixture. Thus, the mediators used do not seem to extend the substrate range for this laccase under these experimental conditions.

Introduction

Marasmius quercophilus is a predominant basidiomycete in typical Mediterranean ecosystems such as evergreen (Quercus ilex L.) or cork oak (Quercus suber L.) litters. This white-rot fungus has been implicated in lignin biodegradation and thus plays an important ecological role since it contributes to the carbon recycling. Phenoloxidases, i.e. laccases, peroxidases or tyrosinases are the enzymatic systems which degrade lignin [START_REF] Tagger | Phenoloxidases of the white-rot fungus Marasmius quercophilus isolated from an evergreen oak litter (Quercus ilex L.)[END_REF][START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white rot fungus Marasmius quercophilus[END_REF]. We have studied different laccases (EC 1.10.3.2.) produced by certain M. quercophilus strains collected in various areas in the south of France [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF][START_REF] Farnet | Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus ilex L.)[END_REF]. These studies attempt to determine whether the biochemical characteristics and the aromatic compound degradation potential of such a polymorphic enzymatic system may vary depending on different parameters: ecosystem, fungal strain or culture conditions. Finding a laccase which has a strong abil-ity to transform aromatic compounds is an important aim for biotechnological applications. Laccases have indeed aroused considerable interest in the last few years because of the reaction they catalyze. They reduce oxygen to water while oxidizing various phenolic compounds in a non-specific radical-based reaction. One of the applications is the degradation of chlorophenols which have been used extensively as pesticides and thus are now widely spread pollutants.

In this study, we described a new isoform of laccase produced by a M. quercophilus strain isolated from evergreen oak litter and which has been induced with ferulic acid. We also compared its biochemical characteristics with those of another isoform also induced with ferulic acid but produced by a M. quercophilus strain isolated from a cork oak litter. Furthermore, this new isoform has been tested on different phenolic compounds both with and without two mediators: 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT), which have been extensively used to extend the range of substrates that can be oxidized and also to enhance the transformation of aromatic compounds [START_REF] Bourbonnais | Electrochemical analysis of the interactions of laccase mediators with lignin model compounds[END_REF].

Materials and methods

Isolation of the strain

M. quercophilus strain 19 was isolated from a part of the site of La Gardiole de Rians, Var, France (1 ha) using the rhizomorphic form of the fungus. A fungal cap culture was firstly made on a malt-agar medium, 20 g l -1 (Bio Mérieux, Marcy l'Etoile, France) and chloramphenicol, 50 mg l -1 (Sigma). Secondly, the pure mycelial culture obtained was used to inoculate an agar medium (whole wheat flour, 20 g l -1 ) which favours rhizomorph production. Then one rhizomorph was used to inoculate a malt-agar plate to obtain a pure dicaryotic culture.

Culture conditions

Precultures were performed in 200 ml Erlenmeyer flasks containing 50 ml of malt extract medium (20 g l -1 ) in static conditions at 28 • C. They were inoculated with a plug of agar cultures (15 g l -1 ) with malt extract (20 g l -1 ). These liquid precultures were used to inoculate two 3000-ml Erlenmeyer flasks with 800 ml of malt extract medium (20 g l -1 ); Tween 80, 0.05%; CuSO 4 , 5 mg l -1 ; ferulic acid, 500 mg l -1 . They were incubated at 28 • C for 12 days under an axial shaking. Enzyme activity was measured by following the oxidation of syringaldazine [N,N -bis-(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine] that leads to its quinone (ε M : 6.5 10 4 M -1 cm -1 ) at 525 nm [START_REF] Harkin | Use of syringaldazine for detection of laccase in sporophores of wood rotting fungi[END_REF] on a spectrophotometer Kontron Uvikon 860. The assay contained 100 l of the culture medium, 2.5 ml of phosphate buffer 0.1 M, pH 5.7 and 10 l of syringaldazine 0.6% (w/v), diluted in methanol. The blank consisted of 100 l of the culture medium and 2.5 ml of the same phosphate buffer.

Purification of the two laccase isozymes

Cultures were filtered on a glass microfiber filter GF/D, 2.7 m (Whatman, England). Then, the filtered culture medium was concentrated in dialysis tubes rated at 12 kDa (32 mm diameter, Poly Labo) using polyethyleneglycol. The samples from the different tubes were pooled using a phosphate buffer 20 mM, pH 5.7 in a final volume of 15 ml. This sample was then loaded on a Mono Q column. A step gradient system of NaCl in Tris-HCl buffer 10 mM pH 6 (0, 0.2, 0.4, 1 M) was used to elute active fractions. Laccase activity was detected using syringaldazine. The active fractions eluted as one peak were pooled and concentrated on an ultrafiltration cell, Model 8200 (Amicon, Beverly, USA) till the volume reached 5 ml. The membrane used (Amicon), was rated at 10 MW (10 kDa) cut-off. This membrane was rinsed with an additional volume of 1 ml of acetate buffer, 0.1 M, pH 4 to remove any bound laccases from its surface.

Effects of pH and temperature on laccase activity

Laccase activity was detected as previously described using 15 l of the purified fraction. Acetate buffer 0.1 M was used for pH range from 4 to 5 and phosphate buffer 0.1 M for pH range from 5.7 to 8. The optimum temperature for laccase activity was determined using 15 l of the purified fraction in acetate buffer 0.1 M pH 4.5. Seven temperatures were tested: 30, 40, 50, 60, 70, 80, 90 • C. The thermal stability of laccase activity was studied using the temperatures mentioned above and after several incubation times (1, 2, 3, 5, 7, 9, 24 and 36 h). Laccase activity was also measured using 15 l of the purified fraction.

Effects of inhibitors on laccase activity

Laccase activity was measured using 15 l of the purified enzyme in acetate buffer 0.1 M, pH 4.5 with ABTS 100 M as the substrate. The inhibitors used at a final concentration of 1 mM were: sodium azide, EDTA, cystein, and sodium diethyldithiocarbamate (DDC). All the chemical products were purchased from Sigma.

Determination of kinetic parameters

Syringaldazine and ABTS were used as substrates for the determination of the V m and K m which were calculated using the Lineweaver-Burk transformation of Michaelis-Menten equation. The concentrations ranged from 0.5 to 50 M and from 1 to 10 M for ABTS and syringaldazine respectively in acetate buffer 0.1 M pH 4.5.

Electrophoresis analysis

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) were carried out according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 4% stacking gel and 12% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Bio-Rad). Molecular weight of the laccase isozyme was determined using high range molecular weight prestained standard (Bio-Rad). Protein band was revealed using the Coomassie blue standard method. For laccase activity staining, acetate buffer, 0.1 M, pH 4.5, with syringaldazine 0.1% or p-phenylenediamine 0.1% was used as the staining solution.

HPLC analysis of aromatic compound degradation

HPLC system was equipped with a C18 column (Merck, 4.6 mm × 250 mm) in the following gradient system: solvent A, water/0.1% trifluoro acetic acid/methanol 90/10 (v/v), solvent B water/0.1% trifluoro acetic acid/methanol 95/5 (v/v), gradient = 0-30 min A 100% to B 100%, 30-40 min B 100% (flow rate 1 ml min -1 ). The aromatic compound degradation was assayed using a reaction mixture (10 ml) containing ABTS or HBT 1 mM, aromatic compound 1000 mg l -1 and 5 U of laccase in acetate buffer 0.1 M pH 4.5. The aromatic compounds used were: catechin, ellagic acid, ferulic acid, gallic acid, protocatecuic acid, p-hydroxybenzoic acid, vanillic acid, veratryl alcohol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP). All the aromatic compounds were purchased from Sigma. The same reaction was performed without mediators. Controls were also realized using only ABTS or HBT with laccase and using ABTS or HBT with the aromatic compound but without laccase. Samples were collected at 0, 2, 4 and 48 h and 30 l of each sample was injected in the HPLC system. P-hydroxybenzoic acid was used as reference in each injection.

Results

The production of the induced isoform was observed on SDS-PAGE after a 5-day incubation and the maximum global laccase activity was obtained after a 12-day incubation (Fig. 1). The laccase isozymes in the culture supernatant were separated by SDS-PAGE: three isoforms were stained with p-phenylenediamine as previously described [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white rot fungus Marasmius quercophilus[END_REF]. The isoform with the lowest molecular weight was then purified using a Mono Q chromatography column (Fig. 2). This purification step allowed us to obtain a single band on the gel using syringaldazine as the substrate. Under denaturing conditions we also observed a major protein band with a molecular weight of about 60 kDa (Scheme 1).

The effect of pH (values ranging from 3 to 8) was examined on laccase activity. The pH optimum of the induced laccase was 4 using ABTS and 4.5 using syringaldazine (Fig. 3). The maximum activity was obtained at 80 • C with these two substrates. The thermal stability of this enzyme was greatest at 30 and 40 • C (100% of the maximal activity remained after a 24 h incubation) while the enzymatic activity quickly decreased (after a 1-h incubation) however high temperatures tested (50, 60, 70, 80, 90 • C). Furthermore, we observed an increase in enzymatic activity from 1250 to 2330 U l -1 for 30 • C and from 1300 to 2700 U l -1 for 40 • C. Fig. 1. Time course of total laccase activity (U l -1 ) in liquid cultures with malt extract medium (20 g l -1 ), Tween 80 (0.05%), CuSO 4 (5 mg l -1 ) and ferulic acid (500 mg l -1 ) at 28 • C. All the standard deviations were below 3%. The first values (1250 and 1300 U l -1 ) are the basal laccase activities at room temperature. The kinetic parameters V m and K m were 2 mM min -1 , 4.2 M for syringaldazine and 3.3 mM min -1 , 11.3 M for ABTS. Laccase activity was not affected by EDTA or cystein as inhibitors while strong inhibition was observed with azide. With DDC, first a decrease in absorption was observed for 11 min, followed by an increase for four more minutes and the absorption finally stabilized for the last 15 min of the experiment (Fig. 4).

All the natural aromatic compounds, gallic acid, tannic acid, protocatecuic acid and vanillic acid were transformed with or without the addition of mediators. For gallic acid, ferulic acid tannic acid or vanillic acid, several new peaks were obtained (Fig. 5) while the transformation of protocatecuic acid led to a single major peak. All these newly transformed compounds eluted after a longer elution time than the initial peaks.

The transformation of chlorophenols was efficient for 2-CP, 2,4-DP and 2,4,6-TCP. A decrease of about 50% of the initial absorption was observed for 2-CP with the enzyme alone and with 2,4-DP with the enzyme added Scheme 1. The profile of the purified laccase induced with ferulic acid from strain 19 on a SDS-PAGE (low molecular weight markers (kDa) on the left line). Fig. 3. The effect of pH on the induced laccase activity (U l -1 ) using ABTS () or syringaldazine (᭡). All the standard deviations were below 3%. with ABTS. Furthermore, with 2,4,6-TCP the decrease in initial absorption was stronger (about 23%) with or without mediators. Moreover, for these three chlorophenols, we detected several peaks which eluted after longer retention times. We did not observe the transformation of pen-Fig. 5. The analysis of the transformation of vanillic acid with the induced laccase using a RP C18 column: ()vanillic acid alone, (

) vanillic acid added with the induced laccase. tachlorophenol whatever the experimental conditions used (Fig. 6).

Discussion

Previous studies have allowed us to purify various isoforms of laccases produced by strains isolated from evergreen oak [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF] or cork oak litter [START_REF] Farnet | Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus ilex L.)[END_REF]. We have shown that the strains isolated from evergreen oak litter exhibited similar laccase patterns under ferulic acid induction: three isoforms were observed [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white rot fungus Marasmius quercophilus[END_REF]. We have already purified and described the two laccase isoforms with higher molecular weights [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF]. In this study we wanted to characterize the third laccase isoform induced with ferulic acid from a M. quercophilus strain isolated from a Q. ilex L. litter. A similar experiment of induction with ferulic acid [START_REF] Farnet | Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus ilex L.)[END_REF] has been realized with a M. quercophilus strain collected from a cork oak litter (Q. suber L.). The biochemical characteristics of this two laccase isoenzymes induced with ferulic acid differ in various ways: optimum pH, thermal stability and V m and K m values (Table 1). The optimum pH for laccase activities is usually low, ranging from 3.0 to 4.5 [START_REF] Heinzkill | Characterization of laccases from a wood-rotting fungi (family Coprinaceae)[END_REF] but great differences have been observed between the two isoforms using ABTS and syringaldazine as the substrates. Furthermore, stability at 40 • C was significantly greater for the laccase from strain 19 (48 h instead of 7 h). Decreases in thermal stability of laccases at higher temperatures have already been described in several studies [START_REF] Nyanhongo | Production of laccase by a newly isolated strain of Trametes modesta[END_REF]. The optimum temperature found (80 • C) was similar for both laccases from strains 19 and C7. Laccases can exhibit very different optimum temperatures from 30 • C for example with the hyphomycete Chalara paradoxa [START_REF] Robles | Characterisation of laccase activity produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH 32[END_REF] to 80 • C with basidiomycete PM1 [START_REF] Coll | Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic basidiomycete PM1 (CECT 2971)[END_REF]. The V m and K m values differed considerably from one isoform to another. The values found for strain 19 were close to those observed for other laccases from white-rot fungi such as Trametes pubescens [START_REF] Galhaup | Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions[END_REF] or Ganoderma lucidum [START_REF] Ko | Purifiaction and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum[END_REF]. We also observed that EDTA and cystein did not inhibit laccase from strain 19 although the absence of inhibition was shown only when EDTA was added to the laccase from strain C7. Furthermore, the staining of SDS-PAGE with syringaldazine was possible, which may suggest the absence of inhibition of laccase with SDS for the concentration used. These properties are particularly interesting since other studies have demonstrated a loss of activity of this enzyme with similar inhibitor concentrations when EDTA, cystein or SDS were used as the inhibiting agents. When compared to the other isoforms of laccases previously purified [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF], biochemical differences were also pointed out: those isoforms exhibited different optimum pHs for laccase activity with syringaldazine as the substrate (pH 5; pH 5 and 6) and also a greater thermal stability of laccase activities. Thus, this study shows the great variations of the biochemical properties of laccases produced by a single fungal species. The transformation with laccase of most of the natural aromatic compounds tested showed several new peaks with hydrophobic properties (longer elution times). This suggests a polymerization of these compounds which was probably due to the formation of radicals by the laccase. Few studies have sought to identify the by-products of laccase transformation. Recently, Carunchio et al. [START_REF] Carunchio | Oxidation of ferulic acid by laccase: identification of the products and inhibitory effects of some peptides[END_REF] have described dimerization of ferulic acid with a laccase. In this study, the authors have identified three intermediates which react to form six different dimers. They separated two major by-products of oligomerization, which allowed them to identify these two compounds using mass spectrometry. It seems that the laccase from M. quercophilus forms many different compounds from gallic acid, ferulic acid and tannic acid. This result is observed with or without mediators thus these non-specific reactions are not due to the action of oxidized mediators alone. The radicals formed with laccase seem to interact in different ways, leading to various polymerized compounds.

The transformed chlorophenols tested have chlorine atoms in ortho or para position. The meta-substituted com-pounds have been found not to be easily transformed [START_REF] Filazzola | Effect of various pollutants and soil-like constituents on laccase from Cerrena unicolor[END_REF]. Indeed, dechlorination of halogenated compounds has been reported with laccase in ortho or para positions with respect to the hydroxyl group of the same ring [START_REF] Schultz | Dehalogenation of chlorinated hydroxybiphenyls by fungal laccase[END_REF]. In this study, the transformation of chlorophenols has shown that the order of reactivity of the induced laccase was 2,4,6-TCP > 2,4-DCP > 2-CP. This is in correlation with the study of Itoh et al. [START_REF] Itoh | Phenolic acids affect transformations of chlorophenols by a Coriolus versicolor laccase[END_REF] which have described the action of a laccase towards several different chlorophenols. Thus, the reactivity of laccase increases with the number of chlorine on the cycle. This may be better explained by the increase in proton acidity of the hydroxyl group because of the chlorine atoms than by a higher specificity of laccase for this chemical compound. The formation of the radical may be favored by the electroattractive property of the chlorine atoms. With chloroaromatic, we have also observed new several peaks at the end of the elution gradient that suggests an oligomerization as described by Leontievsky et al. [START_REF] Leontievsky | Transformation of 2,4,6-trochlorophenol by free and immobilized fungal laccase[END_REF]. For PCP, Aiken and Logan [START_REF] Aiken | Degradation of pentachlorophenol by the white-rot fungus Phanerochaete chrysosporium grown in ammonium lignosulfonate media[END_REF] have reported its biodegradation with the white-rot fungus Phanerochaete chrysosporium. Nevertheless, few studies have described its transformation by white-rot fungus polyphenoloxidases.

In further studies, we will attempt to assess the transformation of aromatic compounds using the supernatant of a culture from M. quercophilus. This would allowed us to evaluate the potential effect of other enzymes such as peroxidases which are also produced by this fungus. The synergistic action of both phenoloxidases may improve the biotransformation of such recalcitrant compounds.

Fig. 2 .

 2 Fig. 2. Purification profile of the laccase induced with ferulic acid from strain 19 using a step gradient of NaCl: (×) laccase activity (U l -1 ), () absorption at 280 nm. The arrow indicates the peak of the induced isoform.

Fig. 4 .

 4 Fig. 4. The effect of inhibitors on the production of the quinone of syringaldazine at 525 nm: ( ) without inhibitor, () with EDTA, (-) with Cystein, (᭹) with sodium azide, (᭡) with DDC. All the standard deviations were below 3%.

Fig. 6 .

 6 Fig. 6. Transformation of 2-CP, 2,4-DCP and 2,4,6-TCP through time. The results are expressed as the percentage of remaining absorption of the initial peak at 280 nm: () 2-CP; (᭡) 2,4-DCP; (᭹) 2,4,6-TCP.

Table 1

 1 The biochemical features of the laccases induced with ferulic acid from strains C7 and 19 of the white-rot fungus Marasmius quercophilus

		Strain C7	Strain 19
	Molecular weight (kDa)	65	60
	pH ABTS	2.6	4
	pH syringaldazine	6.2	4.5
	V m ABTS (M min -1 )	1.1 a	3.3
	K m ABTS (mM)	7.7	113 × 10 -3
	V m syringaldazine (M min -1 )	7.8 × 10 -2	2
	K m syringaldazine (mM)	50 a	4.2 × 10 -3
	Optimum temperature ( • C)	80 a	80
	Thermal stability at 40 • C	After 7 h a	After 48 h
	(50% activity remaining)
	No inhibition	EDTA a	EDTA, Cystein
		SDS a	SDS
	a Data from Farnet et al. [4].