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Laboratoire de Microbiologie, Service 452, U.M.R. CNRS 6116, Institut Méditerranéen d’Ecologie et de Paléoécologie, Faculté des Sciences et Techniques de Saint Jérôme, F-13397, Marseille, Cedex 20, France
J.M. Savoie
Unité de Recherche sur les Champignons, INRA, BP 81, F-33883, Villenave d’Ornon Cedex, France
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ABSTRACT |
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Trichoderma spp., soil filamentous fungi, are antagonists that can cause great losses in mushroom production. We have investigated the influence of T. longibrachiatum on the production of lignocellulolytic enzymes by Pleurotus ostreatus during its vegetative growth on a straw-based cultivation substrate that either had been sterilized, pasteurized or not heat treated. The variations in the lignocellulolytic activities and the electrophoretic patterns in single and dual cultures were used as a tool for perturbation assessment. The various heat treatments of the wheat straw before inoculation affected both the bacterial populations and the abilities of T. longibrachiatum and P. ostreatus to colonize the substrate and to produce extracellar lignocellulolytic enzymes. Interactions between T. longibrachiatum and the microflora of the substrate led to a great decrease of hydrolytic activities due to reduced colonization of the substrate. Pleurotus ostreatus also was affected but it was less sensitive than T. longibrachiatum. As a consequence, in dual cultures with P. ostreatus, the competitive ability of T. longibrachiatum was reduced by bacteria in the substrates. The presence of total microflora or thermotolerant microflora increased the production of phenoloxidase activities by P. ostreatus, despite reduced colonization of the substrate. This contributed to the improvement of the competitive ability of P. ostreatus in the pasteurized substrate. Furthermore, a direct effect of bacteria on T. longibrachiatum also was observed. In sterilized substrate, both laccase and Mn-peroxydase activities were increased dramatically in dual cultures due to a faster production of a laccase isoform, which was stimulated by T. longibrachiatum.
Key words: cellulases, green mold, laccases, mushrooms, peroxydase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Mycelial growth of Pleurotus spp. is fast, and various lignocellulosic waste products can be used as culture substrate (Yildiz et al 2002). The aim of commercial mushroom substrate preparation is to produce a substrate that is optimal and selective for vegetative mycelial growth. In the case of A. bisporus, the white button mushroom, this is accomplished largely by microbial activities during composting. In the case of Pleurotus spp., a wood-rot fungus, this is achieved by the application of various heat treatments to eliminate competitive fungi. Trichoderma spp., soil filamentous fungi, are antagonists that can cause extensive losses in mushroom production (Badham 1991, Jandaik and Guleria 1999). These fungi produce several enzymes involved in degradation of the fungal cell walls that may contain chitinases and glucanases (Sivan and Chet 1989, Geremia et al 1993, Ait-Lahssen et al 2001).
In this study, we investigated the influence of Trichoderma longibrachiatum on the production of lignocellulolytic enzymes by Pleurotus ostreatus during its vegetative growth in a straw-based cultivation substrate. Variations in the production of these enzymes might reveal some effects of T. longibrachiatum because they are involved in the degradation of lignocellulosic materials; reduction of these enzymes indeed might affect the growth of P. ostreatus. Futher-more, the study of these variations might reveal the enzymatic systems that play a role in the response to environmental stress. We focused this analysis on cellulase, ß-glucosidase and on two phenoloxidases, laccase and Mn-peroxidase. Phenoloxidases are enzymes involved in lignin degradation (Hiroi and Eriksson 1976, Thurston 1994). Laccases (EC 1.10.3.2) are blue copper oxidases that catalyse the oxidation of aromatic compounds while reducing oxygen to water. They are relatively nonspecific enzymes that can oxidize monophenol, o- and p-diphenol and aminophenol. Laccase isoforms vary between species and within species (Farnet et al 1999), depending on culture conditions. Mn-peroxidases oxidize phenolic compounds in the presence of H2O2 and manganese. Furthermore, these enzymes have been found to be involved in the response to environmental stress (Rayner et al 1994). Thus the variations of the activities and of the electrophoretic patterns of these enzymes when P. ostreatus was cultured in dual cultures with T. longibrachiatum were used as a tool for perturbation assessment.
We also have investigated the role of the substrate microflora in these antagonistic interactions. Little is known about the influence of bacteria on edible fungus cultures. Thus this parameter was important to consider because this analysis might determine microflora that can inhibit fungal-antagonistic effects. To determine this role we used different types of cultivation substrate: wheat straw without heat treatment, sterilized wheat straw and pasteurized wheat straw.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cultures in wheat straw. – Rye grain was boiled in water and sterilized by autoclaving twice at 121 C for 1 h with incubation at room temperature for 48 h between the two cycles. Bags containing 300 g of moist grains (65% H2O) were inoculated with 1 cm2 of MEA medium with P. ostreatus and placed at 25 C in the dark for 28 d. Before use as inoculum for wheat straw, the spawn were stored at 4 C for 7 d and conditioned at 25 C for 2 d. Wheat straw was shredded into 4–6 cm segments and soaked in water 24 h at room temperature. After leaching, 2% (w/w) of gypsum and 4% of Millichamp® (supplement containing 7.3% organic nitrogen) were mixed with the straw and 90 plastic bags with microporous filters (van Leer, U.K.) were filled with 1 kg of the mixture before application of one of these heat treatments: (i) sterilization by autoclaving twice at 121 C for 1 h with incubation at room temperature for 48 h between the two cycles (30 bags); (ii) pasteurization at 65 C and 98% relative humidity for 24 h (30 bags); (iii) incubation at room temperature for 48 h (30 bags). Nine blocks of 1 kg substrate from each heat treatment were inoculated with P. ostreatus only; nine blocks were inoculated with T. longibrachiatum only; nine blocks were inoculated with both fungi; and nine blocks were not inoculated. Pleurotus ostreatus was inoculated under sterile conditions with 30 g of grain spawn by mixing the spawn and the substrate in the bag to obtain a homogeneous mixture. Trichoderma longibrachiatum inoculum was obtained from sporulating precultures on maltagar medium. Fifty cm2 of these cultures were minced and placed on the wheat straw in the bags. The blocks were incubated at 25 ± 1 C with a 10 h/14 h light/dark cycle for 12 d.
Sampling. – The sampling times were 4, 8 and 12 d after inoculation. Three blocks of each heat treatment were collected as three replications. After estimations of the relative surfaces colonized by P. ostreatus and T. longibrachiatum, the bags were removed and the content of each was homogenized by vigorous mixing in a basket. Four hundred g subsamples from each homogenized bag were used in subsequent analysis and cut into <1 cm segments. The three replications of the treatment combinations (heat treatment x inoculation), were mixed together to obtain a mean sample for immediately counting fungi and bacteria and for lyophilization before the measurement of lignocellulosic enzyme activities.
Substrate colonization. – Zones of the blocks colonized by T. longibrachiatum were identified as dark areas with green spores and marked on the bags. The zones colonized by P. ostreatus were identified as clear areas where the white mycelium was visible and marked on the bags. A grid was superposed on the blocks and percent surface area of each fungus on each block was calculated.
Number of bacterial and fungal colony forming units. – Suspension and dilution technique was used for fungi and total and fluorescent bacterial colony counts in the pasteurized samples and samples that were not heat treated. Samples of 20 g (wet weight) of substrate were blended twice for 10 s in 180 mL of a dispersing solution containing 1.2 g Bactopeptone (Difco) and 6 g tetra-sodium diphosphate decahydrate per liter. Serial dilutions were obtained with this suspension and plated on King’s B medium (Proteose-peptone 2%, K2HPO4 0.15%, MgSO4 0.15%, glycerol 1%, agar 1.5% plus cycloheximide 9 µM), or malt extract (Cristomalt 2%, agar 1.5%, plus antibiotics). After incubation at 25 C, the bacterial and fungal colonies were counted and the quantity of fluorescent bacteria colonies was recorded under UV light (Wood’s lamp). The results were expressed as colony forming units (CFU) per gram of fresh substrate and were the means of at least three replicates.
Enzyme extraction. –
Lyophilized substrate and mycelia samples were used to extract extracellular enzymes. The extraction was performed according to Criquet et al (1999) using 10 g of milled lyophilized substrate in a 1 liter flask containing 200 mL of an extraction solution (Polyvinylpolypyrolidone [Euromedex] 5.7 g, CaCl2 0.2 M, Tween 80 0.05%). These samples were subjected to axial shaking for 1 h at 120 rpm at room temperature. Solids were eliminated by filtration through nylon screen, and filtrates were centrifuged at 10 000 g for 15 min. The supernatants obtained were filtered twice through Whatman GF/D filters (2.7 µm) and through Whatman GF/C filters (1.7 µm). The supernatants of each extract were dialyzed against a Bis-Tris buffer (20 mM, pH6) and concentrated using polyethyleneglycol to final volume of 10% of the initial volume.
Dry mass determination. – To determine the dry mass of the substrate of culture, the substrate was dried in an oven at 100 C for 24 h.
Enzyme activity measurements. –
Laccase activity was measured at 525 nm on a Kontron Uvikon 860 spectrophotometer by following the oxidation of Syringaldazine (N,N'-bis-[3,5-dimethoxy-4-hydroxybenzylidene]hydrazine) that leads to its quinone (
M : 6.5 x 104 M–1 cm–1). The assay contained 500 µL of concentrated extract, 2.5 mL of phosphate buffer 0.1 M, pH 5.7 and 15 µL of Syringaldazine 0.6 % (w/v), diluted in methanol. The blank consisted of 500 µL of extract concentrated and 2.5 mL of the same phosphate buffer. The results were expressed as µmol of substrate oxidized min–1 g–1 of wheat straw dry mass (U.g–1 DM) at room temperature.
Mn-peroxidase activity was measured using the method of Mata and Savoie (1998) with some modifications. This activity was determined by the oxidation of 3-dimethylaminobenzoic acid (DMAB, 7.5 mM) and 3-methyl-2-benzothia-zoline hydrazone (MBTH, 0.65 mM) at 590 nm (M = 3.29 x104 M–1 cm–1) in a phosphatecitrate buffer 0.1 M, pH 5.0. 250 µL of extract were used and MnSO4 (1 mM) and H2O2 (30% w/v) were added to the reaction mixture. Assays without MnSO4 or H2O2 also were performed to check whether other phenoloxidases can react with the substrates used. The results were expressed as µmol of substrate oxidized min–1 g–1 of wheat straw dry mass (U.g–1 DM) at room temperature.
To measure cellulase activity, 0.1 mL of the extract was incubated at 50 C for 1 h in 0.9 mL of 50 mM acetate buffer (pH 5.0) with 0.1% carboxymethylcellulose (CMC). After incubation, the sugars released from the hydrolysis of CMC were measured in the samples using the colorimetric method of Somogyi-Nelson (Alef and Nannipieri 1995). Cellulase activities were expressed in µmol of glucose released per g–1 of wheat straw dry mass in cultivation substrates for 1 h (µmol glucose. g–1 DM h–1).
ß-glucosidase activity was measured using the Alef and Nannipieri method (1995). The extract (0.05 mL) was incubated at 50 C for 15 min in 0.95 mL of 50 mM acetate buffer (pH 5.0) with 3 mM of p-nitro-phenyl-ß-D-glucopyr- anoside. After incubation, 0.5 mL of a 4% Na2CO3 solution was added and absorbance was measured at 412 nm. Results were expressed as µmol of p-nitrophenol released g–1 of wheat straw dry mass during 1 h (µmol PNP g–1 h–1).
Each measurement of enzyme activities was duplicated.
Electrophoresis analyses. –
Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) were carried out according to Laemmli (1970) using 4% stacking gel and 12% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Bio-Rad).
For laccase electrophoresis, p-phenylenediamine (0.1%) was used as the substrate in phosphate buffer 0.1 M, pH 5.7. In Mn-peroxidase electrophoresis, DMAB and MBTH (0.1 % w/v) were used with MnSO4 and H2O2 in phosphate-citrate buffer 0.1 M, pH 5.0. Controls were performed without MnSO4 and H2O2. To determine cellulase electrophoretic profiles, the gels were made using 1% CMC instead of the distilled water in separating gel. The gels were incubated 5 min in a 0.1% Congo Red solution and the color was removed using NaCl 1 M until clear bands showing the CMC hydrolysis appeared.
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RESULTS |
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). This interval was at the limit of the susceptibility of the suspension-dilution technique. There was little difference in the total number of bacteria among the different treatments. However, no fluorescent bacteria of the genus Pseudomonas were observed in the pasteurized substrates whereas they were abundantly present in substrates that were not heat treated. No mycelial colonies other than T. longibrachiatum and P. ostreatus were observed on the plates.
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). In dual cultures in the autoclaved substrate, T. longibrachiatum greatly reduced colonization by P. ostreatus and colonized one-third of the substrate. In both pasteurized and substrate not heat treated, P. ostreatus colonized two-thirds of the substrate whereas T. longibrachiatum colonized approximately 10% of the substrate.
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). The most activity was observed in the pasteurized substrate. Little activity was measured in the sterilized substrate. However, when both T. longibrachiatum and P. ostreatus were inoculated in the substrate, phenoloxidase activities were higher in both autoclaved and substrates that were not heat treated but not in the pasteurized substrate (FIG. 1).
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).
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). When Pleurotus was cultured with Trichoderma, several bands of laccases were observed on the gel at various times, depending on culture conditions. The electrophoresis profile showed four bands of laccases after 4 d of incubation and two bands after 8 d and 12 d of incubation. In this profile, the isoform with the highest molecular weight (FIG. 3A, arrow) was produced after 8 d of incubation when P. ostreatus was cultivated alone (data not shown) and after 4 d of incubation when P. ostreatus was cultured with T. longibrachiatum. The Mn-peroxidase profiles also showed two isoforms (data not shown).
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, strong bands with arrows) whether P. ostreatus was cultivated alone or in dual cultures with T. longibrachiatum. Two additional weak, high molecular-weight bands were observed on all unsterilized substrates (FIG. 3B, arrows on right). These bands were absent on all sterilized substrates, suggesting their origin from bacteria. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The presence of bacteria and the differences in bacterial communities in the cultivation substrate associated with the heat treatments affected both the mycelial colonization and to a lesser extent the level of CM-cellulase and ß-glucosidase production by T. longibrachiatum. Interactions between T. longibrachiatum and the microflora of the substrate led to a decrease of hydrolytic activities, probably due to a reduced colonization of the substrate. These results are in agreement with other studies where the inhibitory effects of some bacteria having antifungal properties were the main factor affecting the potential of Trichoderma spp. in ecosystem colonization (de Boer et al 1998, Savoie et al 2001a). Bacterial strains can inhibit the growth of Trichoderma by production of volatile organic compounds (Mackie and Wheatley 1999) or by releasing antibiotics (Nielsen et al 2000). These compounds can provoke cell perturbations at the membrane structure level (Vanittanakom and Loeffler 1986). Pseudomonas spp. have been identified as antagonists of Trichoderma spp. (Upadhyay et al 1991, Ellis et al 2000).
Pleurotus ostreatus also was affected by the presence of some bacteria in its cultivation substrate as suggested by the greatest laccase and Mn-peroxydase activities of Pleurotus when cultivated on pasteurized substrate compared to that sterilized. Lang et al (1997) have observed both higher substrate mineralization and colonization where Pleurotus spp. was cultured with soil microflora. In the presence of bacteria P. ostreatus may react in various ways. One of these was observed by Tsuneda and Thorn (1994) in dual cultures of Pseudomonas and Lentinula edodes where fungal lytic enzymes degraded the bacterial wall. However, a positive effect on mycelial growth of P. ostreatus induced by the presence of Pseudomonas fluorescens recently has been reported (Cho et al 2002). A great decrease of laccase activity also was observed on pasteurized substrate when Pleurotus was cultivated with Trichoderma after 12 d of incubation. This result is difficult to explain without laccase activities at longer incubation times. This would have let us describe the evolution of such activities. Furthermore, laccase activities hardly can be compared to Mn-peroxidase activities, which are very weak. Thus we have not been able to make a global assessment of phenoloxidase activities through time.
In the present work, the capacity of T. longibrachiatum to compete in dual cultures was decreased in presence of other microorganisms in the substrates. The antagonistic effect of the bacteria from mushroom substrates has been observed previously (Savoie et al 2001a) and this may be used by mushroom growers for cultivating wood-rot mushrooms under nonsterile conditions.
The presence of total microflora or thermotolerant microflora (the bacteria that grow on the substrate after pasteurization) increased the production of phenoloxidases by P. ostreatus despite a less abundant colonization of the substrate. The production of laccases already has been described as a response to environmental stresses (Rayner et al 1994, Score et al 1997, Savoie et al 1998). We already have reported that a preliminary adaptation of Pleurotus spp. to Trichoderma spp. metabolites led to the induction of laccase production, which let Pleurotus compete efficiently against the antagonist (Savoie et al 2001b, Savoie and Mata 2002). The stimulation of laccase activities in the pasteurized substrate might contribute to the improvement of the capacity of P. ostreatus to compete in the pasteurized substrate in addition to the direct effect of bacteria on T. longibrachiatum. This might be because polyphenoloxidases are enzymes catalyzing reactions of oxidation that produce radicals. These radicals are highly reactive chemical compounds, which can lead to membrane perturbations.
In sterilized substrate, both laccase and Mn-peroxydase activities were increased in dual cultures. Savoie et al (2001b) have reported an increase of laccase activity in vitro, in dual cultures of P. ostreatus and T. longibrachiatum, but Mn-peroxydases were not studied. The present in situ study is also in agreement with similar data obtained with Lentinula edodes cultivated in wheat straw (Savoie and Mata 1999).
The variability of isoenzyme production in fungal antagonisms has not been explored in situ. Few data are available on the effect of fungal antagonism on enzyme production kinetics and also on the potential production of new induced isoforms (Savoie et al 1998). Most of these studies concerned global activity measurements or fungal interactions on solid culture media. However, the present study shows that a laccase isoform was produced faster when P. ostreatus was cultivated with T. longibrachiatum.
In this study we demonstrated the involvement of phenoloxidases in the response of P. ostreatus to both fungal and bacterial antagonism. This analysis may be extended to other enzymatic systems in oxydo-reduction metabolism to assess their importance in fungal dual cultures. Furthermore, this study has shown a role of the microflora of the culture substrate in fungal interactions. In further studies, the bacteria involved in these phenomena should be identified. Thereafter, we should attempt to improve the development in cultivation substrates of bacterial strains that both could inhibit Trichoderma spp. growth and promote edible mushroom development, thus producing better yields.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: amfarnet{at}netscape.net
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LITERATURE CITED |
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) sterilization, (
) pasteurization, (
) without heat treatement. All standard deviations were below 3%.
