Fungal Biology

Manuscript Draft
Manuscript Number:
Title: Do storage conditions of the spawn affect the capacity of colonization of a wheat-straw based
substrate by Agaricus subrufescens?
Article Type: Original Research
Keywords: lignocellulolytic enzymes; solid-state NMR of 13C; almond mushroom; Agaricus blazei;
Agaricus brasiliensis
Abstract: Storage conditions of the spawn of edible fungi are of major importance since this facilitates
the cultivation and the production of mushrooms. Here we determined whether standard storage
conditions at 10C or 15C, were suitable for the tropical species Agaricus subrufescens which is
affected by the temperature of 2-4C commonly used for Agaricus bisporus. We assessed the potential
of the spawn to colonize and transform horse manure and wheat-straw-based mushroom compost
produced for A. bisporus during the spawn running phase. Lignocellulolytic activities and substrate
transformation (using CP/MAS NMR of 13C) were used as microbiological and chemical markers and
the functional diversity of the substrate microbial communities were also estimated. Storage of the
spawn has an effect on this capacity of colonization: in certain cases, it enhanced lignocellulolytic
activities and substrate transformation as described by the aromaticity ratio and a humification ratio
calculated from NMR data. This result indicates that mycelium growth probably occurred during
storage at 10 or 15C leading to a larger amount of biomass in the inoculum. Moreover, the diversity of
the microbial community of the substrate was also favored in certain cases showing that the electivity
of the substrate was maintained. Thus these findings indicate that recommendations for the mushroom
producers can be established for A. subrufescens cultivation under European standard conditions.

Highlights (for review)

Spawn of Agaricus subrufescens stored at 10 or 15 C colonises substrate effectively

Storage conditions can even enhance the colonisation potential depending on the strain
Markers of substrate transformation are higher than those obtained with A. bisporus

Manuscript
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Do storage conditions of the spawn affect the capacity of colonization of a wheat-straw

based substrate by Agaricus subrufescens?

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Anne-Marie Farnet*1, Leila Qasemian1, Frdrique Peter-Valence1, Florence Ruaudel1, Jean-

Michel Savoie2, Sevastianos Roussos1, Isabelle Gaime-Perraud1, Fabio Ziarelli3and Elise

Ferr1

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Mditerranen de Biodiversit et dEcologie marine et continentale, Aix Marseille Universit,

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Campus de lEtoile, 13397, Cedex 20, Marseille, France, *anne-marie.farnet@imbe.fr, Tel/Fax

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+33 4 91 28 81 90

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dOrnon.

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Equipe Vulnrabilit des systmes microbiens, IMBE, UMR. CNRS. IRD. 7263, Institut

INRA, UR1264 MycSA, Mycologie et Scurit des Aliments, BP 81, F- 33883, Villenave

TRACES Campus de lEtoile, 13397, Aix-Marseille Universit, Marseille, Cedex 20, France

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Abstract

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Storage conditions of the spawn of edible fungi are of major importance since this facilitates

27

the cultivation and the production of mushrooms. Here we determined whether standard

28

storage conditions at 10C or 15C, were suitable for the tropical species Agaricus

29

subrufescens which is affected by the temperature of 2-4C commonly used for Agaricus

30

bisporus. We assessed the potential of the spawn to colonize and transform horse manure

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and wheat-straw-based mushroom compost produced for A. bisporus during the spawn

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running phase. Lignocellulolytic activities and substrate transformation (using CP/MAS NMR

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of 13C) were used as microbiological and chemical markers and the functional diversity of the

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substrate microbial communities were also estimated. Storage of the spawn has an effect on

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this capacity of colonization: in certain cases, it enhanced lignocellulolytic activities and

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substrate transformation as described by the aromaticity ratio and a humification ratio

37

calculated from NMR data. This result indicates that mycelium growth probably occurred

38

during storage at 10 or 15C leading to a larger amount of biomass in the inoculum.

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Moreover, the diversity of the microbial community of the substrate was also favored in

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certain cases showing that the electivity of the substrate was maintained. Thus these

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findings indicate that recommendations for the mushroom producers can be established for

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A. subrufescens cultivation under European standard conditions.

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Key words: lignocellulolytic enzymes, solid-state NMR of 13C, almond mushroom, Agaricus

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blazei, Agaricus brasiliensis.

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Introduction

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Mushroom cultivation is an important agricultural domain which combines the production of

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nutraceuticals with organoleptic and potentially medicinal properties using a

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biotechnological process which recycles agricultural by products. For all these reasons,

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production of Agaricus species has been widely developed and, in European countries, the

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button mushroom A. bisporus is the main cultivated species.

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However, an emerging Agaricus species, A. subrufescens Peck, also named A. blazei Murrill

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sensu Heinemann, A. rufotegulis Nauta or A. brasiliensis Wasser, M. Didukh, Amazonas &

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Stamets (Kerrigan 2005), is actively cultivated in Brazil since the early 1990s, first in the Sao

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Paolo state and then extended to other regions close to the Atlantic coast. It had become an

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important export production for this country. This species exhibits an almond flavor

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probably because of benzaldehyde and benzyl alcohol (Stijve et al., 2003), which can be of

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great value as foods or food flavoring materials. Moreover, edible mushrooms are known to

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have bioactive compounds in their biomass, such as polysaccharides with anti-cancer or

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tumor suppressive activity, and extracts from the basidiocarp of this species have been

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shown to reduce tumor size in rats (Jumes et al., 2010).

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Interestingly, A. subrufescens can be cultivated on the composts originally made for the

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cultivation of the button mushroom (Largeteau et al., 2011). The inoculum used for the

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culture in compost is made of mycelium grown under sterile conditions on cereal grains and

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is known as the spawn. The type of material used for the spawn (wheat, rye or millet grains

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) and its preparation have already been described as a crucial parameter in mushroom
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cultivation (Mamiro et al., 2008). During this key step the fungus fully colonizes the solid

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carrier chosen, starts to produce lignocellulolytic enzymes and thus fungal biomass is

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obtained in large amount to further inoculate the culture substrate (Ruiz-Rodriguez et al.,

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2011). The spawn is generally produced by specific companies and send to mushroom

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growers, which implies storage for several weeks before use. Unfortunately, A. subrufescens

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is suffering damage when exposed for prolonged periods at temperatures of 4 C or lower

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(Kerrigan 2005). Consequently, mushroom growers have difficulties for maintaining spawn

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under refrigeration for long time periods without loss of mycelium viability and spawn-

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makers recommend to store it at temperatures around 12 C. However there is no

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information on the consequences of storages at these temperatures on the quality of the

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spawn and the further ability of mycelium to colonize cultivation substrates. In order to

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thoroughly define the process which could be recommended for its cultivation, we

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investigated whether spawn of A. subrufescens produced on rye seeds stored at 10 or 15C

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for 15 days or 1 month was affected in its substrate colonizing potential of horse manure

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and wheat-straw based compost. The substrate colonizing potential of several strains was

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tested using microbiological and chemical markers: i) both lignocellulolytic activities i.e.

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laccases and cellulases, ii) functional diversity of bacterial communities of the substrates

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using a catabolic level physiological profile (CLPP) and iii) the chemical transformation of

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lignocellulosic material using 13C solid-state NMR spectroscopy. Here, functional diversity of

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microbial communities was investigated since this microbial part of the substrate is essential

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for mushroom production under solid-state fermentation conditions. It is thus important to

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focus on substrate microbial communities since they are involved in both substrate

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transformation and selectivity. We also included in this study an A. bisporus strain commonly

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cultivated in France as control of substrate colonization.

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Material and Methods

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The fungal strains

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Six strains of A. subrufescens and one strain of A. bisporus from the Collection of

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Germplasms of Agaricus in Bordeaux (CGAB), INRA, were used. CA454 is a subculture of the

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collection strain WC837 in PSUMCC registered as A. blazei and assumed to be similar to

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ATCC 76739, which is claimed to be a copy of the original strain cultivated for the first time

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in Brazil in the 1960s . CA646 and CA647 are two cultivars sold in Europe by Mycelia

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(Belgium) under the reference numbers 7700 and 7703 respectively. CA560 was isolated in

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Botucatu, Brazil (1999) from a commercial farm and CA572 is a Brazilian cultivar purchased

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by D.C. Zied in 2007, both strains have been proved to be genetically different (Foulongne-

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Oriol et al., 2012). Their original reference numbers are ABL-99/28 and ABL-07/58

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respectively, in the Culture Collection of the Edible and Medicinal Mushroom Module of So

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Paulo State University (UNESP). An A. bisporus strain, Bs527, for which storage is commonly

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performed at 2-4C was used as control. Bs527 was a copy of the cultivar 30A (Euromycel)

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placed in the CGAB in 1997.

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Mesocosm experimental set-up

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Precultures were performed in glass Petri dishes containing 12 g dry weight of cooked and

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coated rye grains (spawn substrate used by spawn makers, provided by Somycel, Langeais,
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France) inoculated with 6 plugs (1x1 cm) of malt agar cultures. They were incubated in a

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climatic chamber at 25C and 90 % RH (relative humidity). Then, for controls, they were

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directly used to inoculate the mesocosms while, for assays, these precultures were

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incubated in a climatic chamber either at 10C or 15 C for 15 days or 1 month and were

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used immediately after storage to inoculate the mesocosms. These mesocosms were

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prepared with 450 g of fresh mushroom compost (135 g dry weight) placed in a 2 L glass pot

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in order to obtain a final volume of compost of 20*16*6 cm. They were inoculated with 3%

122

(w/w) of rye grains (dry weight), which were thoroughly mixed with the compost under

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aseptic conditions. The compost was commercial compost produced by SARL Renaud et Fils

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at Avy, France, for the cultivation of A. bisporus. The main raw materials used were horse

125

manure and wheat straw, and the phase I of composting was performed indoor. The quality

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of the compost was assessed by the yields obtained in commercial cultivation facilities that

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reached standards. Three mesocosms were prepared for each strain and were incubated in a

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climatic chamber at 25C and 90 % of RH for 15 days in darkness. An un-inoculated control

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(with the compost alone), UIC, was also prepared and incubated under the same conditions.

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Measurement of the substrate colonizing potential

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The substrate transformation potential of the fungal strains was tested by monitoring i) both

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lignocellulolytic activities produced by the different strains ii) the functional diversity of

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bacterial communities of the substrates using a catabolic level physiological profile (CLPP).

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iii) the chemical transformation of lignocellulosic material using 13C solid-state NMR

136

spectroscopy.

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Lignocellulolytic activity assays

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For each mesocosm, enzyme extraction was performed as previously described by Velazquez

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et al., (2004) using 10 g of substrate in a 1 L flask containing 200 mL of an extraction solution

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(Polyvinylpolypyrolidone 5.7 g, CaCl2, 0.2M, Tween 80, 0.05%). These samples were

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subjected to axial shaking for 1 hour at 120 rpm at room temperature. Solids were

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eliminated by centrifugation at 10 000 g for 15 min and filtration through Whatman GF/D

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filters (2.7 m) and through Whatman GF/C filters (1.7 m). The filtrates of each extract

144

were concentrated using polyethyleneglycol to a final volume of 10% of initial volume.

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Laccase activities were measured by monitoring the oxidation of syringaldazine to quinone

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(M= 65 000 M-1.cm-1) at 525 nm in acetate buffer, 0.1 M, pH 5 on a spectrophotometer

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Biomate 3 (Fischer Bioblock Scientific). One unit (U) of laccase activity is defined as the

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amount of moles of quinone of syringaldazine produced per min and per mL of sample at

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room temperature. Three assays were performed for each enzyme extract.

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CM-cellulase activities were measured with carboxymethylcellulose (CMC) and using a

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simplified methodology adapted from that of Somogyi-Nelson as described by Farnet et al.

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(2010). Solution A was composed of 50 mL of CuSO4, 2 %, Na2SO4, 4 g, complemented with

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50 mL of a solution of Na2CO3, 25 g, NaHCO3, 20 g and sodium potassium tartrate, 25 g,

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q.s.p. 1 L. Solution B was composed of ammonium-molybdate, 25 g, H2SO4, 21 ml, and

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Na2HAsO4, 7H2O, 3 g, q.s.p. 450 mL. To measure CM-cellulase activity, 0.1 mL of the extract

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was incubated at 50 C for 1 h in 0.9 mL of 50 mM acetate buffer (pH 5.0) with 0.1% CMC. 1

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mL of solution A was added to 1mL of the reaction medium and 1 mL of distilled water; then

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the mixture was boiled for 20 minutes and cooled down in a cold water bath for 15 minutes.

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1 ml of solution B was added and the mixture was left for 50 minutes at room temperature

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and then centrifuged for 2 minutes at 10 000 g. The absorption was measured at 870 nm.
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Dilutions were performed when necessary. For the calibration curves, 1 mL of solution A was

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added to 1 mL, 750 L, 500 L, 250 L of a solution of glucose (80 mg/L) q.s. 1mL. Three

163

assays were performed for each enzyme extract.

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Microbial catabolic profiles via Biolog EcoPlate

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For each mesocosm, one gram of substrate was vigorously shaken in 100 mL of a sterilized

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desorption-solution (Sodium pyrophosphate 0.08 g/L) for 1h and then brought to a final

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OD=0.001 at 595 nm. 150 L of the extracted solution were used to inoculate all 96 wells of

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a BIOLOG EcoPlate (Biolog, California, USA). One EcoPlate was used for three mesocosm

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extracts since each 96-well plate containing three replicates, each with 31 different carbon

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sources, one per well, and a water blank. The plates were incubated at 25C for 48h and the

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absorbance was read at 590 nm using a microplate reader (Tecan, France). To measure the

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functional diversity (FD), the Shannons diversity index (Shannon and Weaver, 1949) was

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calculated (Zack et al., 1994) from:

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FD = - pi(ln pi), where pi is the ratio of color development of well i to the sum of color

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development of all positive wells. Microbial activity for each microplate was assessed as

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average well-color development (AWCD) calculated as follows: AWCD = OD i / 31 where

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OD i is the optical density for each well.

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The Cross-Polarization Magic Angle Spinning 13C Nuclear Magnetic Resonance (CP/MAS 13C

181

NMR) procedure

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CP/MAS 13C NMR spectra were obtained on a Bruker DSX 400 MHz spectrophotometer

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operating at 100.7 MHz. Samples (600 mg) were spun at 10 KHz at the magic angle. Contact
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times of 2 ms were applied with a pulse width of 2.8 s and a recycle delay of 3 s. Chemical

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shift values were referenced to glycine signal (carbonyl C at 176.03 ppm). The 13C NMR

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spectra were divided into seven chemical- shift regions, according to Ziarelli (2004). Dmfit

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2003 software was used to determine the intensity of each chemical-shift-region (Massiot et

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al., 2002). An index of decomposition (HR#1 Alkyl-C / O-Alkyl-C) was calculated as described

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by Baldock et al. (1997). An aromaticity ratio was also calculated to estimate the degree of

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humification of organic matter and Alkyl-C/COOH-C ratio HR#2 was used to assess organic

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matter transformation using the alkyl chain length of organic acids.

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Statistical analysis

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The significance of differences (enzyme activities, AWCD and H from Biolog, NMR ratio)

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between means of three experiments was tested by nonparametric multiple comparison of

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Kruskal-Wallis. Each experiment was considered as an independent sample. Pearson

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correlation was tested between enzyme activities and chemical groups defined by 13C NMR

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and ratios calculated from these data.

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The optical absorbances of the 30 wells in the ECO plate were subjected to Principal

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Component Analysis (PCA) after 48h of incubation, considering each substrate as a variable.

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The intensities of the 22 peaks obtained via CP/MAS NMR of 13C analysis of the culture

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substrate were used for PCA. StatistiCAVs 6 (StatSoft, Maison-Alfort, France) was used for

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statistical analysis and P-value<0.05 is considered as significant.

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Results

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Lignocellulolytic activities

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To asses to which extent the mycelium of the strains studied were able to colonize the

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substrate after different storage treatments, we focused on the main enzyme systems

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involved in lignocellulosic material transformation, which have been proved to be efficient as

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microbiological marker of substrate colonization . For lignocellulolytic activities, storage of

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the spawn had an effect on cellulase and laccase activities measured after incubation of

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spawned compost for 15 days at 25C (Figures 1 and 2) though no global trend can be clearly

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observed. After certain storage conditions, these activities were indeed enhanced. More

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precisely, cellulase activities were significantly higher than in the controls for strains CA646

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and CA560 stored at 10C and for and for strain CA572 stored for 1 month at 10 or 15 C

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(Figure 1). Only weak differences were observed with the controls for strains CA647, CA454

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and CA646 (in this latter case, the lower values concerned storage at 15C only). For A.

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bisporus, cellulase activities remained similar except for storages at 10 C for 1 month, which

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were higher than those in control. Concerning laccase activities (Figure 2), lower values but

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with weak differences were found for certain strains (strains CA560 and CA572 and the A.

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bisporus strain) but in most of the cases, storage had no effect or led to higher laccase

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activities (strains CA454, CA646 and CA647). In non- inoculated compost, no laccase and

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cellulase activities were detected.

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Catabolic Physiological Profile of microbial communities of the substrate using BIOLOG

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EcoPlate.

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We further investigated whether spawn storage at 10 or 15 C may have an indirect effect on

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the microbial communities of the substrate by modifying the potential of substrate

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colonization by the mycelium: this was assessed by the catabolic physiological profile of the

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substrate microorganisms. Global microbial activity was measured by Average Well Color

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Development (AWCD) and functional diversity via the Shannon-Weaver Index, H. AWCD was

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similar than in control in most of the treatments. When AWCD varied from the control,

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higher values were observed for certain strains and only in substrate inoculated with spawn

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previously stored at either 10 C or 15C (Figure 3). For functional diversity characterized by

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H, the same result was observed: higher H than in control was found when spawns of

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certain strains had been stored at 10C or 15C (Figure 4). These differences in both AWCD

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and H were observed for A. bisporus Bs527 and strains CA560, CA647 and CA454. For strain

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CA646, no changes were found compared to control and a lower value in both AWCD and H

240

was observed with CA572 only. Figure 5 shows the result of PCA analysis using optical

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densities of all the substrates used as variables. Axes F1 and F2 accounted for 59.39 and

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10.42 % of the variance respectively. This figure shows that the temperature used for

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storage (10 or 15 C) did not globally affect microbial catabolic diversity since the projections

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were not clearly differentiated on the PCA (squares vs triangles). On the other hand, the

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time of storage (15 days or 1 month) influenced this diversity (black vs white) mainly

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according to axe F2, as observed for strains CA647 and CA560, and to a lesser extent

247

according to F1, for strains CA572 and CA646. In the case of strain CA647, the microbial

248

communities of the substrate were more strongly modified by the different storage

249

conditions: control for this strain was clearly differentiated from the projections

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corresponding to the different pretreatments. Three treatments with A. bisporus and CA560

251

stored for 15 days at 15C were located in the vicinity of UIC, showing no significant changes

252

in the microbial community.

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Organic matter transformation of the substrate via solid-state NMR of 13C

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Different ratios were calculated from the NMR spectra to determine how storage conditions

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of the spawn may affect the capacity of transformation of the substrate: an aromaticity ratio

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(AR) and two Humification ratios (HR1 = Alkyl-C/ O-Alkyl-C and HR2 = Alkyl-C/ COOH-C). For

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AR, when an effect was observed, it was always a higher ratio in the case of substrate

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colonized by spawn previously stored at 10 or 15C than in control (Table 1). The same

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tendency was observed for HR1. Higher values in both AR and HR1 indicate that the

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recalcitrant fraction of organic matter (linked to Alkyl-C and Aromatic-C signals) accumulated

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in the substrate, while O-Alkyl-C signal, assigned to polysaccharides, decreased. For HR2, the

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higher ratio in substrate inoculated with pre-incubated spawn than in control showed that

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long-alkyl chain acids were predominantly observed.

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Discussion

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Agaricus subrufescens is known to be sensitive to low temperatures and this particular

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characteristic is an issue to define adequate storage conditions (De Mendoca et al., 2005).

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This fungal species grows at higher optimal temperatures than those of A. bisporus: for

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instance Colauto et al (2008) found that optimal temperatures for mycelial growth was

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around 30 C depending on the strains studied; De Mendoca et al (2005) reported optimal

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temperatures ranging from 25 to 28 C and Llarena et al. (2013), optimal temperatures

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varying from 26 to 30 C, with similar range of variation for cultivars and wild strains. A long-

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time storage at low temperatures such as 4 C is known to be lethal for A. subrufescens (Zou,

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2005). Mycelial growth rate decreased gradually over storage time at 4C of spawn on
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sorghum grains used for long term storage of genetic resources (Mata and Savoie, 2012).

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Thus, ideal storage conditions, limiting consequences on the ability to colonize cultivation

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substrate, were still to be defined. Here we found that storage at 10 or 15 C for a period as

279

long as 1 month, did not strongly affect the potential of different A. subrufescens strains in

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their ability of early colonization and transformation of the compost used as cultivation

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substrate during the spawn running period. Though this global trend was observed,

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responses to the different storage conditions varied with the A. subrufescens strain studied.

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In our previous study (Farnet et al., 2013), we have observed different levels of

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production of lignocellulolytic enzymes depending on the A. subrufescens strains under

285

study. Ruiz-Rodriguez et al. (2011) have also found that production of ligninolytic enzymes

286

(i.e. peroxidases and laccases) varied with the Pleurotus pulmonarius and P. ostreatus strains

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considered, when cultivated on a wheat straw- based substrate complemented with olive

288

mill waste. Laccase activity in compost colonized by A. bisporus is known to be mainly

289

produced by this fungus and can be used as a marker of mycelial biomass production, at

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least during the exponential phase of growth (Wood 1979). For instance Savoie (1998) have

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observed that, after a lag phase, laccase activity in compost inoculated with A. bisporus

292

became significant at day 10 and increased until day 29, prior to primordia formation. In our

293

study, A. bisporus strain Bs527 had the lower activities observed for all storage treatments

294

compared to control, showing that this strain was affected in its ability to produce mycelial

295

biomass during spawn-running. On the other hand, most activities measured with A.

296

subrufescens were higher than those with A. bisporus and for certain treatments they were

297

higher than in non-stored controls. This positive effect of storage at cold temperatures on

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the biomass production during spawn-running was observed with other parameters.
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In comparison to laccase activities, in previous studies with A. bisporus (Savoie, 1998)

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cellulase activities fell of 38 % between 0 and 10 d and reached a plateau at less than 10 % of

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the initial value whereas the activities in non-spawned plots were stable or increased during

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the same period. This might be due to the replacement of the initial microbial community of

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the compost by a community influenced by the mycelium of the mushroom after spawning.

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In the present study, compost was stored for several days before being used and no cellulase

305

activity was detected in non-inoculated compost. Cellulase activities in fresh compost were

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actually probably due to residual activities of thermophilic microorganisms, which developed

307

during composting. Consequently, here, cellulase activities observed after incubation for 15

308

days had most probably been produced by A. subrufescens and new mesophyll

309

microorganisms which have grown during incubation at 25C.

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To thoroughly investigate the consequences of mycelium storage on A. subrufescens ability

312

to colonise compost and change its biotic environment, the diversity of microbial

313

communities of the substrate was also considered via their functional structure. We found

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that storage of the spawn had probably an effect on A. subrufescens mycelium, which

315

consequently influenced the microbial communities during mycelial colonization of compost,

316

by enhancing both their global catabolic activity, and thus their potential to transform the

317

substrate, and their biodiversity. Ecoplates are known to focus on the functional diversity of

318

the bacterial communities since the reduction of tetrazolium dye cannot be accomplished by

319

fungi (Heuer and Smalla, 1997, Preston-Mafham et al., 2002). Thus the Agaricus strain, which

320

colonises the culture substrate, is not supposed to be involved in the catabolic potential

321

measured here. Here, the high diversity of microbial communities of the substrate is of
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major importance in the selectivity of the mushroom culture substrate since it prevents from

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the colonization of potential antagonists (Velazquez et al., 2008). PCA was used to give

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additional information about how the functional diversity of these communities varied.

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Though the effects of both time and temperature of storage were observed, the more

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drastic conditions i.e. 10 C and 1 month did not lead to a strong variation in diversity of the

327

substrate microbial communities according to the PCA. For instance, for strains CA572,

328

CA454, projections of the control were close to those after storage at 10C for 1 month -

329

CA572 (C4) and CA454 (C4) - indicating that the catabolic profile is similar to that of the

330

control. Thus, storage at 10C for 1 month did not have the strongest effect compared to the

331

other storage conditions (projection close to control). Colonisation of the substrate by the

332

mycelium of A. subrufescens may have modified the associated microbial communities

333

because of microbial interactions including competition for nutrients or production of

334

antagonistic metabolites. Here we found that the diversity index, H, remained constant or

335

was even higher after spawn storage and PCA added complementary information by

336

indicating that for certain strain, this diversity actually changed compared to control (i.e.

337

different carbon sources were used depending on storage).

338

We also monitored the dynamics of organic matter transformation in the substrate using

339

solid-state NMR of 13C. Both AR and HR1 ratios are supposed to increase when the easily-

340

biodegradable fraction of organic matter, i.e. mainly polysaccharides, is effectively

341

transformed.

342

For HR2, our results indicated that the amount of acids with long aliphatic-chains increased

343

in the substrate between control and certain storage conditions. These molecules can be

344

linked to phospholipids from cell membranes (Peter-Valence et al., 2011) and this shows that
15

345

mycelial fungal biomass is more extensively produced under these conditions i.e. when the

346

inoculum is previously incubated at 10 or 15 C. This results obtained with solid-state NMR

347

of 13C corroborated those found with laccase activities. Storage of the spawn at 10 or 15C

348

indeed led to an increase in the potential for colonization of the culture substrate and a

349

greater production of mycelium during spawn-running. This can also be linked with the

350

efficient polysaccharide transformation shown by the increase in both the AR and HR1

351

ratios.

352

A. subrufescens can be considered as an important edible mushroom because of its

353

medicinal and culinary properties, which culture conditions still have to be precisely

354

described. In our previous study (Farnet et al.,2013) we found that a horse manure and

355

wheat-straw-based compost produced under physico-chemical conditions commonly used

356

for the button mushroom A. bisporus was suitable for this tropical Agaricus species. Here,

357

our investigations focused on storage conditions of the spawn, which is a very important

358

parameter to favour mushroom cultivation. Our study revealed that storage conditions of

359

the spawn for up to 1 month, at relatively low temperatures for this fungus, did not further

360

affect substrate colonisation by this species. Storage under certain conditions even improved

361

its potential of colonization and this potential seems to be strain-dependent. These results

362

are encouraging since they demonstrate that these storage conditions at 10 to 15 C cannot

363

be ruled out in the cultivation process. However, further studies should determine whether

364

these storage conditions have effects on crop yields or mushroom quality in order to confirm

365

these first findings.

366
367

Acknowledgements
16

368

This work was supported by the French ANR, project ANR-09-BLAN-0391. We are very

369

grateful to Mrs Marjorie Sweetko for her very helpful assistance in English language.

370
371

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Zou X, 2005. Effect of Zn supplementation on the growth, amino acid composition,

444

polysaccharide yields and anti-tumour activity of Agaricus brasiliensis. World Journal of

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Microbiology and Biotechnology 21:261264.

446
447

Figure captions

448
449

Fig. 1 Cellulase activities measured in a horse manure- and straw-based compost inoculated

450

with Agaricus bisporus (Bs527) or six strains of A. subrufescens and incubated at 25C for 14

451

days. The spawn was previously inoculated at 15C for 15 days , 15 for 1 month, 10C for 15

452

days or 10C for 1 month or not. The letters define the mean values which are statistically

453

different from each other according to multiple comparison of Kruskal-Wallis (Dunn

454

Procedure)

455
456

Fig. 2 Laccase activities measured in a horse manure- and straw-based compost inoculated

457

with Agaricus bisporus (Bs527) or six strains of A. subrufescens and incubated at 25C for 14
20

458

days. The spawn was previously inoculated at 15C for 15 days, 15 for 1 month, 10C for 15

459

days or 10C for 1 month or not. The letters define the mean values which are statistically

460

different from each other according to multiple comparison of Kruskal-Wallis (Dunn

461

Procedure)

462
463

Fig. 3 AWCD (Average Well Color Development) based on Ecoplate data after 48h of

464

incubation of the microbial extract from a horse manure- and straw-based compost

465

inoculated with Agaricus bisporus (Bs527) or six strains of A. subrufescens and incubated at

466

25C for 14 days. The spawn was previously inoculated at 15C for 15 days, 15 for 1 month,

467

10C for 15 days or 10C for 1 month or not. The letters define the mean values which are

468

statistically different from each other according to multiple comparison of Kruskal-Wallis

469

(Dunn Procedure)

470
471

Fig. 4 Shannon Weaver index, H, based on Ecoplate data after 48h of incubation of the

472

microbial extract from a horse manure- and straw-based compost inoculated with Agaricus

473

bisporus (Bs527) or six strains of A. subrufescens and incubated at 25C for 14 days. The

474

spawn was previously inoculated at 15C for 15 days, 15 for 1 month, 10C for 15 days or

475

10C for 1 month or not. The letters define the mean values which are statistically different

476

from each other according to multiple comparison of Kruskal-Wallis (Dunn Procedure)

477
478

Fig. 5 PCA (based on Pearson correlation) performed using OD at 48h of each Ecoplate

479

carbon source (n=31) as variables. The two first factors (F1+F2) accounted for 72% of total

480

variance. UIS: Un-Inoculated substrate, horse manure- and straw-based compost inoculated
21

481

with Agaricus bisporus (strain Bs527) or six strains of A. subrufescens, and incubated at 25C

482

for 14 days. The spawn was previously inoculated at 15C for 15 days (C1, ), 15 for 1

483

month (C2,), 10C for 15 days (C3, ) or 10C for 1 month (C4 ) or not (C, )

484
485
486

22

Table(s)

Table 1 Aromaticity ratio 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 13C NMR data from horse manure and straw based substrate inoculated with six different Agaricus subrufescens strains and A.
bisporus, and incubated at 25C for 14 days. The spawn was previously inoculated at 15C for 15 days (C1), 15 for 1 month (C2), 10C for 15 days (C3) or
10C for 1 month (C4) or not (C). The different letters indicate when values differ significantly according to Kruskal-Wallis test and Dunn procedure for
multiple comparisons of means (p < 0.05)

C
C1
C2
C3
C4

Bs 527
0.204
0.200
0.196
0.200
0.208

CA 454
+/- 0.011 ab 0.166
+/- 0.005 ab 0.158
+/- 0.005 a
0.162
+/- 0.006 ab 0.202
+/- 0.004 b
0.178

C
C1
C2
C3
C4

Bs 527
0.413
0.365
0.336
0.374
0.404

CA 454
+/- 0.017 c
0.209
+/- 0.009 ab 0.185
+/- 0.037 a
0.224
+/- 0.020 abc 0.352
+/- 0.017 bc 0.331

+/- 0.011
+/- 0.011
+/- 0.046
+/- 0.044
+/- 0.052

C
C1
C2
C3
C4

Bs 527
2.856
2.819
2.716
2.911
2.973

+/- 0.056 ab
+/- 0.278 ab
+/- 0.105 a
+/- 0.122 ab
+/- 0.118 b

CA 454
1.121
1.720
1.928
2.695
2.541

+/- 0.043
+/- 0.297
+/- 0.125
+/- 0.054
+/- 0.360

+/- 0.013
+/- 0.004
+/- 0.007
+/- 0.007
+/- 0.029

ab
a
ab
b
ab

CA 560
0.196
0.200
0.183
0.175
0.178
CA 560
0.407
0.344
0.349
0.357
0.337

a
ab
ab
b
b

CA 560
2.122
2.450
2.263
2.427
2.125

Aromaticity Ratio
CA 572
+/- 0.019 b 0.155
+/- 0.013 b 0.191
+/- 0.012 ab 0.189
+/- 0.006 a 0.176
+/- 0.008 ab 0.185
HR1
CA 572
+/- 0.060 a 0.302
+/- 0.035 a 0.382
+/- 0.024 a 0.375
+/- 0.032 a 0.323
+/- 0.009 a 0.404
HR2
CA 572
+/- 0.109 a 2.051
+/- 0.116 b 2.409
+/- 0.046 ab 2.420
+/- 0.292 ab 2.082
+/- 0.132 a 2.178

CA 646
+/- 0.007 a
0.170
+/- 0.020 b
0.189
+/- 0.005 b
0.186
+/- 0.006 ab 0.182
+/- 0.010 b
0.180

+/- 0.017
+/- 0.014
+/- 0.005
+/- 0.012
+/- 0.014

a
a
a
a
a

CA 647
0.162
0.195
0.179
0.159
0.187

+/- 0.007
+/- 0.004
+/- 0.011
+/- 0.033
+/- 0.005

a
b
ab
a
ab

+/- 0.062
+/- 0.017
+/- 0.036
+/- 0.058
+/- 0.064

a
a
a
a
a

+/- 0.136
+/- 0.022
+/- 0.197
+/- 0.381
+/- 0.137

a
b
b
ab
ab

CA 646
+/- 0.011 a
0.370
+/- 0.011 bc 0.348
+/- 0.018 abc 0.407
+/- 0.028 ab 0.399
+/- 0.034 c
0.355

+/- 0.012
+/- 0.043
+/- 0.075
+/- 0.026
+/- 0.064

a
a
a
a
a

CA 647
0.361
0.380
0.372
0.314
0.395

CA 646
2.220
2.454
2.359
2.113
2.199

+/- 0.222
+/- 0.144
+/- 0.243
+/- 0.311
+/- 0.359

a
a
a
a
a

CA 647
1.930
2.602
2.592
2.437
2.414

+/- 0.207 a
+/- 0.153 b
+/- 0.231 b
+/- 0.080 ab
+/- 0.209 ab

Figure(s)

Strain CA560

10

12

cd

8
6

bc

ab

Cellulase activity
(mol glucose/g DW/h)

Cellulase activity
(mol glucose/g DW/h)

12

Strain CA572

8
6

ab

control 10C
15d

10C
1m

15C
15d

15C
1m

control 10C
15d

Strain CA646
cd

ab

bc
d

2
0

Cellulase activity
(mol glucose/g DW/h)

10

15C
15d

15C
1m

12

Strain CA647

10
8
6

4
2

ab

control

10C
15d

10C
1m

15C
15d

Experimental conditions
12

15C
1m

Strain CA454

8
6
4

bc

cd

10C
1m

15C
15d

10C
1m

15C
15d

15C
1m

Experimental conditions

10

control 10C
15d

ab

Cellulase activity
(mole glucose/g DW/h)

Cellulase activity
(mol glucose/g DW/h)

12

10C
1m

Experimental conditions

Experimental conditions

Cellulase activity
(mol glucose/g DW/h)

bc

10

12

Strain Bs527

10
8
6
4

b
a

2
0

control 10C
15d

Experimental conditions

15C
1m

control 10C
15d

10C 1
m

15C
15d

Experimental conditions

15C
1m

Figure(s)

cd

100

ab

control 10C
15d

10C
1m

50

bc

Laccase activitiy
(mol/g DW/h)

Laccase activitiy
(mol/g DW/h)

150

200

Strain CA560

200

Strain CA572

100

15C
1m

control 10C
15d

200

Strain CA646

150

ab
a

50
10C 1
m

15C
15d

100

ab

50

a
control

Laccase activity
(mol/g DW/h)

c
b

10C
15d

10C
1m

15C
15d

15C
1m

Strain Bs527

200

Experimental conditions

150

15C
1m

15C
1m

Strain CA454

100

bc

Experimental conditions
200

15C
15d

Strain CA647

150

0
control 10C
15d

10C
1m

Experimental conditions

Laccase activity
(mol/g DW/h)

Laccase activity
(mol/g DW/h)

200

Laccase activity
(mol/g DW/h)

0
15C
15d

Experimental conditions

50

50

100

150

150
100

50

ab

bc

0
control 10C
15d

10C
1m

15C
15d

15C
1m

Experimental conditions

control 10C 10C 1 15C

15d
m
15d

15C
1m

Experimental conditions

Figure(s)

CA560

1.2

1.2

0.2

0.2
0

1.2

15C
15d

15C
1M

10C
1M

0.4

0.4

0.2

0.2

1.2

15C
1M

bc CA454

10C
15d

bc

ab

15C
15d

1.2

10C
15d

10C
1M

0.2

0.2

0
15C
1M

15C
1M

Bs527
bc

bc

ab

0.4

0.4

15C
15d

ab

0.6

0.8

0.6

10C
1M

ab

10C
1M

10C
15d

CA647

0.8
0.6

15C
15d

15C
1M

0.6

15C
15d

1.2

0.8

10C
15d

CA646

0.8

ab

0.4

abc

0.6

0.6
0.4

bc

1
0.8

ab

0.8

CA572

10C
15d

10C
1M

a
C

15C
15d

15C
1M

10C
15d

10C
1M

Figure(s)

1.5

CA560

1.5

ab

CA572

15C
15d

15C
1M

ab
1

0.5

0.5

0
C

1.5

15C
15d

15C
1M

10C
15d

10C
1M

CA646
a

1.5

0.5

0.5

10C
15d

10C
1M

CA647
bc

bc

bc

15C
1M

10C
15d

10C
1M

ab
1.5

15C
15d

15C
1M

CA454
ab

10C
15d

10C
1M

ab

Bs527

1.0

0.5

0.5

15C
15d

15C
1M

1.5

0.0
10C
15d

10C
1M

15C
15d

ab

0
C

15C
15d

15C
1M

10C
15d

10C
1M

Figure(s)