Mycologia, 95(2), 2003, pp. 191-199.
© 2003 by The Mycological Society of America
Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism
Jean-Michel Savoie 1
Unité de Recherche sur les Champignons, INRA, BP 81, F-33883 Villenave d'Ornon Cedex, France
Gerardo Mata
Departamento Hongos, Instituto de Ecologia, A. C., Apdo. Postal 63, C.P. 91000, Xalapa, Veracruz, Mexico
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ABSTRACT
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Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma®, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.
Key words: Agaricus, green mold, laccases, Lentinula, Pleurotus
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INTRODUCTION
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Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms such as Lentinula edodes (Berk.) Pegler, and Pleurotus spp., and which cause wood rot. In the past 15 yr, new aggressive biotypes have led to severe crop losses in Agaricus bisporus (Lange) Imbach, a leaf-litter rot (Ospina-Giraldo et al 1999
). These biotypes, previously named Trichoderma harzianum Th2 in Europe and Th4 in America, recently have been renamed Trichoderma aggressivum (Samuels et al 2002). They are strong antagonists of mushrooms (Savoie et al 2001b) and are adapted for growth in Agaricus mushroom compost by resisting the inhibiting effects of bacteria in this cultivation substrate (Savoie et al 2001a). No defense reaction of A. bisporus cultivars to T. aggressivum attack has been observed (Mamoun et al 2000, Mumpini et al 1998). Production of emergent hyphae, brown-line formation and production of laccases are reactions of L. edodes, Pleurotus eryngii and Pleurotus ostreatus to confrontations with mycelia of T. harzianum or T. aggressivum, or with their extracellular metabolites (Savoie et al 2001b). That contributes to the ability of the mushroom to resist Trichoderma spp. This reaction also is seen during cultivation of L. edodes on lignocellulosic substrates (Savoie and Mata 1999).
The competitive ability of L. edodes was improved by modifying the composition of substrates used for inoculum growth, and consequently the incidence of Trichoderma spp. was reduced (Mata et al 1998). One of the modifications was the addition of components rich in lignin and phenols, which could act as inducers of laccases and of the overall defense system (Savoie et al 2000).
The aims of this study were to establish whether improvement of mushroom resistance to T. aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent.
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MATERIALS AND METHODS
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Fungi –
Twenty-nine French wild strains of Agaricus bisporus (Table I), 29 strains of Lentinula edodes from different origins (Table II) and 18 strains of Pleurotus spp. (Table III) were used in this study. A. bisporus isolates were all A. bisporus var. bisporus, with average spore numbers between 2.0 and 2.5, as defined by Callac et al (1993). They all had cream to brown caps with pilei color having L values between 60 and 85 (Moquet et al 1997). All were from The Institut National de la Recherche Agronomique/Centre Technique du Champignon collection, CGAB, France. Most L. edodes strains were cultivars used in different countries to produce the Shiitake mushroom. They were both from the Institut National de la Recherche Agronomique collection, BxC (France), and the Instituto de Ecologia collection, IE (Mexico). Among Pleurotus strains, three were P. djamor, six were P. ostreatus and nine were P. pulmonarius. They were from the Instituto de Ecologia collection.
A cultivated strain of A. bisporus, with the code Bs 26 in the CGAB collection, and a P. ostreatus strain (BxC-JMO95) also were used in a specific experiment.
The strain of Trichoderma aggressivum f. europaeum, previously named T. harzianum biotype Th2, isolate B, was isolated from A. bisporus cultivation compost (Mamoun et al 2000).
The strains were stored on agar media at 4 C in the collections.
Media –
YME (0.2% yeast extract, 2% malt extract) or YMEA (+1.5% agar) and Cristomalt (2%) were used as basic media. Lysing Enzyme from T. harzianum (Sigma®, L1412) is a lyophilized powder obtained from cultures of T. harzianum. This product contains about 80% protein, with cellulase, chitinase and protease activities. Supplementation of YMEA with Lysing Enzymes was made by diluting the product in water (0.75 g 10 mL-1) and sterilizing with a filter (0.22 µm). This solution was added to 1 L of autoclaved media. In controls not supplemented, 10 mL of sterile water were added. Liquid medium also was supplemented with Lysing Enzymes (final concentration 1 g L-1) or fractions of the Lysing Enzymes solution at concentrations equivalent to 1 g L-1 of Lysing Enzymes. Fractions were obtained by ultrafiltration (10 000 Da) through an ultrafree-PFL low-binding cellulose membrane (Millipore). The components retained by the membrane constituted Fraction 1 and the filtrate was Fraction 2. Fraction 3 was obtained after heating fraction 1 at 80 C for 45 min.
Improvement of resistance to T. aggressivum by adaptation to T. harzianum metabolites –
To determine if mushrooms might be adapted to greater resistance to T. aggressivum by being exposed to extracellular metabolites from T. harzianum, A. bisporus (CGAB-Bs26), L. edodes (BxC-Le27) and P. ostreatus (BxC-JMO95) were cultivated for 9 d at 25 C on YMEA and YMEA supplemented with Lysing Enzymes. The colony diameters were recorded at 5 and 9 d on 20 replicate Petri dishes. Mycelium of T. aggressivum from 2-day-old precultures on YMEA were placed on the margin of mycelial colonies developed by A. bisporus, L. edodes and P. ostreatus, and their reactions were observed for 4 d.
To determine which fraction of Lysing Enzyme was effective on L. edodes to induce laccase production as a reaction and adaptation to the antagonism by T. aggressivum, the strain BxC-Le27 was cultivated at 25 C for 9 d in 30 flasks containing 50 mL of Cristomalt liquid medium. Treatments with Lysing Enzymes and its fractions were performed by adding to the cultures 1 mL of solutions equivalent to 50 mg of Lysing Enzymes. After 8 d at 25 C, the content of each flask was filtered through a preweighted glass-fiber filter and dried at 80 C for 48 h to estimate the mycelial biomass. The filtrates were collected aseptically in sterile flasks. Ten mL were removed and stored at -20 C before being used for measurement of laccase activity and replaced by 10 mL of sterilized malt-extract solution at 50 g L-1. This concentrated malt extract was added to compensate for nutrient depletion due to the growth of L. edodes. Each flask was inoculated with non-sporulated mycelium of T. aggressivum scraped from the surface of one Petri dish containing a 3-day-old culture on YMEA. After 7 d at 25 C, the mycelial biomass of T. aggressivum was measured in the same way as L. edodes. Controls were obtained in the same method but without inoculation of L. edodes at the beginning. Laccase activity in the culture fluids was measured with ABTS as substrate (Savoie et al 2001b).
Effects of T. harzianum metabolites on mycelial growth –
A preliminary culture on YMEA at 25 C was obtained from collection plates for each mushroom strain. Inoculum disks (diam 5 mm) were taken from these precultures and placed in the center of Petri dishes with YMEA supplemented or not with Lysing Enzymes solution. The inoculated media were incubated at 25 C in the dark and the diameters of mycelial colonies were recorded (two perpendicular diameters per colony) every two or three days until they reached the margin of the plates. This first mycelial growth on YMEA or supplemented YMEA was termed Culture 1 (Fig. 1). Culture 2 was obtained with inoculum disks from Culture 1 on YMEA plus Lysing Enzymes. The inoculum disks were taken from the part between the center and the margin of the colonies. Twenty-one-day-old Culture 1 was used for the 29 A. bisporus isolates, 14-day-old Culture 1 was used for the 29 L. edodes isolates and the 18 Pleurotus spp. isolates. Culture 2 was incubated at 25 C in the dark, and the diameters of mycelial colonies were recorded (Fig. 1). Six replicate Petri dishes were inoculated for each strain and each media, in both Culture 1 and Culture 2. Mycelial growth was the slope of the growth curve obtained from mycelial colony diameters at each measurement time.
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FIG. 1. Methodology used for measuring the effects of Lysing Enzymes on mycelial growth rate, and the adaptation of mushrooms to these compounds. The arrows indicate the origin of the inoculum for each culture
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Statistical analysis –
Means of mycelial growth, biomass or laccase activity were analyzed by one-way ANOVA and Fisher's Least-Significant-Difference Test. A hierarchical cluster analysis with single linkage and Euclidean distance was performed on group isolates, according to their ratios of mycelial growth rates on different media: Culture 1 YME + Lysing Enzymes/Culture 1 YME; Culture 2 YME + Lysing Enzymes/Culture 1 YME; Culture 2 YME + Lysing Enzymes/Culture 1 YME + Lysing Enzymes. All analyses were performed with the Systat® package (SPSS Inc., USA).
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RESULTS
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When added to Cristomalt agar, Lysing Enzymes affected the mycelial radial growth of A. bisporus, L. edodes and P. ostreatus (Table IV). The diameters with Lysing Enzymes were 73 to 79% of the values in controls for A. bisporus, whereas the percentages were 88 to 89% for P. ostreatus. Lentinula edodes was slightly affected during the first 5 d of cultures, but the mean diameter at 9 d was significantly higher with Lysing Enzymes than without.
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TABLE IV. Mycelial colony diameter (mm) after cultivation for 5 and 9 d on both YMEA agar and YMEA agar supplemented with Lysing Enzymes from T. harzianum
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Trichoderma aggressivum mycelium was placed in contact with these cultures. Agaricus bisporus did not produce any brown line in the medium but did produce some brown droplets at the mycelium surfaces; T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. aggressivum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on the controls and after only 2 d on the assays. Differences in development of green spores at the surface of the colonies were estimated visually. The production of spores by T. harzianum was more pronounced on YMEA plus Lysing Enzymes than on YMEA.
The improvement in the resistance of a fungus to T. aggressivum was studied in liquid cultures with the L. edodes strain that showed strong adaptation in the first experiment. The biomass production in liquid medium was significantly affected by Lysing Enzymes (Table V). The active fraction was Fraction 1 (compounds with molecular weights higher than 10.000 Da), whereas no significant effect on biomass production was observed in Fraction 2. In Fraction 3—=uivalent to Fraction 1 where enzymes were inactivated by heat treatment—higher biomass was observed (Table V). Otherwise, both Lysing Enzymes and Fraction 1 dramatically increased extracellular laccase activity, with a significantly higher effect for Fraction 1 (Table VI). Trichoderma aggressivum was cultivated in the same media after L. edodes was removed (assays) and in controls without previous culture of L. edodes. Significantly higher biomass was produced in the assays than in the controls with Cristomalt and Cristomalt supplemented with Fraction 2 and Fraction 3, but the difference was not significant with Fraction 1 (Table VII). Biomass production of T. aggressivum in assays was not different in Cristomalt, Cristomalt supplemented with Lysing Enzymes and Cristomalt supplemented with Fraction 1, whereas significantly higher biomass was produced with Fraction 2 or Fraction 3 (Table VII).
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TABLE V. Mycelial biomass produced by Lentinula edodes during incubation for 17 d in Cristomalt liquid media supplemented with fractions of Lysing Enzymes from Trichoderma harzianum
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TABLE VI. Extracellular laccase activity produced by Lentinula edodes measured after incubation for 17 d in Cristomalt liquid media supplemented with fractions of Lysing Enzymes from Trichoderma harzianum
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TABLE VII. Mycelial biomass produced by Trichoderma harzianum during incubation for 7 d in Cristomalt liquid media supplemented with fractions of Lysing Enzymes and previously used for growing Lentinula edodes in the same media without previous growth of L. edodes
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The differences of reaction among the three species, the variability within each species and differences among species of the same genus were studied in a large sample of isolates. For the group of wild isolates of A. bisporus, the mean of mycelial growth on both Culture 1 and Culture 2 with Lysing Enzymes was significantly lower than in culture without Lysing Enzymes (Table VIII). For L. edodes and Pleurotus spp., the mean of mycelial growth on Culture 1 with Lysing Enzymes was significantly lower than on Culture 1 without Lysing Enzymes. The mean in Culture 2 with Lyzing Enzymes was not significantly different from the mean in the other media (Table VIII). A hierarchical cluster analysis was performed to group isolates according to their ratios of mycelial growth on different media (Fig. 2). The maximum Euclidean distance between isolates of L. edodes and Pleurotus spp. was 0.12. Distances as great as 0.62 were observed among A. bisporus isolates. Five A. bisporus isolates grouped with L. edodes and Pleurotus isolates. The isolates Ab 09, Ab 23 and Ab 20 played the role of out-group in the dendrogram. These isolates were characterized by low mycelial growth in controls, and they were greatly affected during the first culture with Lysing Enzymes. Their ratios of second culture to first culture with Lysing Enzymes were actually high (>1.5) for this species but with low absolute values for the mycelial growth rates.
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TABLE VIII. Mycelial growth rate of Agaricus bisporus, Lentinula edodes and Pleurotus spp. on Cristomalt Agar media supplemented with Lysing Enzymes (L.E.) from Trichoderma harzianum
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FIG. 2. Dendrogram derived from cluster analysis (single linkage, Euclidean distance) from three ratios of mycelial growth rate on different media: Culture 1 YME + Lysing Enzymes/Culture 1 YME; Culture 2 YME + Lysing Enzymes/Culture 1 YME; Culture 2 YME + Lysing Enzymes/Culture 1 YME + Lysing Enzymes
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DISCUSSION
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Increases of laccase activity in the interface between wood-degrading fungi, or between Trichoderma sp. and brown-rot basidiomycetes, have been observed (Rayner et al 1994, Score et al 1997). Lentinula edodes was shown to resist Trichoderma spp. attack by producing (1) large amounts of laccases (p-phenol oxidase, EC 1.10.3.2) and (2) barrages characterized by brown lines in the contact zones (Tokimoto and Komatsu 1979). Laccase production was induced by extracellular metabolites produced by T. harzianum (Savoie et al 1998). These extracellular metabolites—defined as the enzymatic fraction produced after treatment of T. harzianum with Lysing Enzymes (Sigma®)—was shown to increase extracellular laccase activity and reduce L. edodes biomass production. Consequently, the culture fluid containing these laccases affected the biomass production by T. aggressivum. Its biomass was not different from that in controls without precultivation of L. edodes, because when it was cultivated in culture fluid of L. edodes without the enzymatic fraction of Lysing Enzymes, its biomass production was stimulated. The antifungal activity of T. harzianum strains used in plant protection involves production of antibiotics including compounds affecting the integrity of fungal membranes and production of such fungal cell wall-degrading enzymes as chitinases, ß-1,3- and ß-1,6-glucanases, proteases and 
-1,3-glucanases (Ait-Lahsen et al 2001). Reaction mechanisms of L. edodes, including production of extracellular laccases, probably are due to cell-wall degrading enzymes produced by T. aggressivum (Savoie et al 2001a), which also are present in Lysing Enzymes from T. harzianum.
Production of emergent hyphae, brown-line formation and production of laccases have been compared during mycelial confrontations of T. harzianum or T. aggressivum isolates with seven wood-rotting and seven leaf-litter-rotting basidiomycetes (Savoie et al 2001b). Lentinula edodes and P. ostreatus reacted strongly, whereas A. bisporus did not react. In this study, the three species were exposed to a solution of T. harzianum extracellular metabolites obtained from Lysing Enzymes, to improve their resistance to T. aggressivum. The mycelial growth of A. bisporus was the most affected by Lysing Enzymes in the culture medium, whereas L. edodes was able to adapt to these metabolites in the culture. As a result, L. edodes or P. ostreatus produced brown lines more rapidly when exposed to T. aggressivum mycelium than in controls without precultures with Lysing Enzymes. This improvement in defense might be considered as an induced resistance. Agaricus bisporus had a high susceptibility to T. aggressivum, and resistance induction was not efficient. This litter-degrading fungus is a mushroom cultivated on compost prepared with cereal straw and horse or poultry manure. In such media, most Trichoderma species have only low colonization abilities due to their susceptibility to antagonistic bacteria (Savoie et al 2001a, b). It is only in the past few years that T. aggressivum have been responsible for dramatic disorders in commercial cultivation. Consequently, the A. bisporus cultivated strain used here was not selected in breeding programs for resisting this antagonist. There is a need for selection of A. bisporus strains with the ability to adapt to Trichoderma extracellular metabolites.
Current and previous observations were from experiments in which only one isolate of each species was studied. To complete this investigation, several isolates of A. bisporus, L. edodes and Pleurotus species were assayed for their ability to adapt to T. harzianum metabolites (Lysing Enzymes), as L. edodes isolate BxC-27 did above. A technique of successive cultivations with chemical inducers was proposed by Mata et al (1997) to study the induction of laccase activities and adaptation of L. edodes to these toxic phenolic compounds. The same system was used here by comparing mycelial growth rates on the first and second culture both with and without Lysing Enzymes. Globally, the wild isolates of A. bisporus were greatly affected by Lysing Enzymes, as was the cultivated strain. For L. edodes and Pleurotus spp., adaptation to Lysing Enzymes was indicated by little difference between the mean growth rates on the second culture with Lysing Enzymes and controls without Lysing Enzymes, whereas the first culture was significantly affected. These observations are in agreement with those obtained with only one isolate in each group, but the variability within each group was different, as reported by the dendrogram derived from cluster analysis (Fig. 2).
There was no relation between the genetic similarities and the adaptation of the isolates to Lysing Enzymes. For example, Le 17, Le 18 and Le 19 were hybrids obtained by crossing Le 02 and Le 03; they did not group. Pl 14 and Pl 15 were hybrids obtained by crossing Pl 12 and Pl 13. Pl 15 was close to Pl 12 on the dendrogram, but Pl 14 did not group with Pl 12 or Pl 13. Three species of Pleurotus from different continents were used. They grouped with L. edodes isolates, and their variability in adaptation to Lysing Enzymes was less important than that of the wild isolates of A. bisporus, from France. Some A. bisporus isolates grouped with L. edodes and Pleurotus spp. isolates. They were those with the higher ratios of mycelial growth in the second culture with Lysing Enzymes to the control without Lysing Enzymes. For these isolates, adaptation resulting in improvement of mycelial growth in second cultures was observed.
By screening a large collection, we can expect to find other wild isolates of A. bisporus with the ability to adapt to Trichoderma extracellular metabolites. Such isolates will be interesting material for breeding programs. Improvement in the resistance to an antagonist by introduction of some of its metabolites to the culture medium is a potential approach to the protection of mushrooms.
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ACKNOWLEDGMENTS
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This work was supported in part by Conseil Régional d'Aquitaine Grant 20010305013 and co-operation program ECOS/ANUIES, M00-A01. We are grateful to Nathalie Minvielle (at INRA) and Rosalía Pérez Merlo (at IE) for excellent technical assistance.
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FOOTNOTES
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1 Corresponding author. savoie{at}bordeaux.inra.fr 
Accepted for publication July 9, 2002.
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LITERATURE CITED
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ABSTRACT
INTRODUCTION
