Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism

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1 Mycologia, 95(2), 2003, pp by The Mycological Society of America, Lawrence, KS Issued 7 May 2003 Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism Jean-Michel Savoie 1 Unité de Recherche sur les Champignons, INRA, BP 81, F Villenave d Ornon Cedex, France Gerardo Mata Departamento Hongos, Instituto de Ecologia, A. C., Apdo. Postal 63, C.P , Xalapa, Veracruz, Mexico Abstract: 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 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. Accepted for publication July 9, Corresponding author. savoie@bordeaux.inra.fr Key words: Agaricus, green mold, laccases, Lentinula, Pleurotus INTRODUCTION 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. MATERIALS AND METHODS Fungi. Twenty-nine French wild strains of Agaricus bisporus (TABLE I), 29 strains of Lentinula edodes from different 191

2 1 MYCOLOGIA TABLE I. List of strains, date of collection, geographic origin and habitat of wild Agaricus bisporus used the study CGAB collection Year isolated Location in France Habitat notes Ab 01 Ab 02 Ab 03 Ab 04 Ab 05 Ab 06 Ab 07 Ab 08 Ab 09 Ab 10 Ab 11 Ab 12 Ab 13 Ab 14 Ab 15 Ab 16 Ab 17 Ab 18 Ab 19 Ab 20 Ab 21 Ab 22 Ab 23 Ab 24 Ab 25 Ab 26 Ab 27 Ab 28 Ab 29 Bs 167 A Bs 419 B Bs 422 A Bs 286 A Bs 72 Bs 78 M Bs 342 Bs 75 A Bs 76 A Bs 437 B Bs 433 A Bs 426 A Bs 433 B Bs 426 D Bs 285 Bs 275 Bs 190 Bs 240 Bs 279 Bs 341B Bs 250 Bs 247 Bs 369 Bs 254 Bs 416 B Bs 118 A Bs 70 D Bs 69 A Bs 85 B Leaf litter beneath Cedrus Composting grass Horse manure Horse manure Leaf litter beneath Picea Composting plant residues, Quercus Leaf litter beneath Chamaecyparis Leaf litter beneath Pinus Composting grass Hay ensilage Horse manure Leaf litter beneath Picea Plant residues 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 and nine were. 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 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.. 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 m). 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. s were obtained by ultrafiltration ( Da) through an ultrafree-pfl low-binding cellulose membrane (Millipore). The components retained by the membrane constituted 1 and the filtrate was 2. 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. harzian-

3 SAVOIE AND MATA: MUSHROOM ADAPTATION TO TRICHODERMA 193 TABLE II. List of strains and origin of Lentinula edodes used the study Le 01 Le 02 Le 03 Le 04 Le 05 Le 06 Le 07 Le 08 Le 09 Le 10 Le 11 Le 12 Le 13 Le 14 Le 15 Le 16 Le 17 Le 18 Le 19 Le 20 Le 21 Le 22 Le 23 Le 24 Le 25 Le 26 Le 27 Le 28 Le 29 collection BxC-Le28 BxC-Le29 BxC-Le33 BxC-Le34 BxC-Le35 BxC-Le36 BxC-Le37 IE 40 IE 105 BxC-Le25 BxC-Le68 BxC-Le63 BxC-Le60 BxC-Le13 BxC-Le17 BxC-Le52 BxC-Le75 BxC-Le79 BxC-Le84 BxC-Le27 IE 208 IE 124 IE 123 IE 171 IE 122 IE 117 IE 112 IE 207 IE 149 Source/origin Cultivars, France (Royal Champignon, S610) Cultivars, France (Somycel, 4055) Cultivars, France (Le Lion, VO84) Cultivars, France (Le Lion, VO122) Cultivars, France (Le Champion, 072) Cultivars, France (Somycel, 4080) Cultivars, France (Somycel, 4004) Hong Kong (from Chang L35) Cultivars, USA (Fungi Perfecti, CS.2) Japan, on a market Japan, on a market Japan (from K. Ono) Japan (from K. Ono) ATCC48861 Unknown Cultivars, USA (Lambert Spawn) Hybrid [BxC-Le29 BxC-Le33] Hybrid [BxC-Le29 BxC-Le33] Hybrid [BxC-Le29 BxC-Le33] Cultivars, France (Royal Champignon, S600) New Zealand, NZFS-210 Cultivars, Guatemala (from R. de León) Cultivars, Guatemala (from R. de León) Netherlands ( CBS ) Cultivars, Guatemala (from R. de León) Korea (from Lee) Cultivars, Japan (Mori P5) Papua New Guinea (from Harvard University, DSH-147PNG) Cultivars, USA TABLE III. List of species/strains and origin of Pleurotus spp. used the study Collection Species Location/Origin Pl 01 Pl 02 Pl 03 Pl 04 Pl 05 Pl 06 Pl 07 Pl 08 Pl 09 Pl 10 Pl 11 Pl 12 Pl 13 Pl 14 Pl 15 Pl 16 Pl 17 Pl 18 IE 121 IE 218 IE 221 IE 131 IE 38 IE 239 IE 49 IE 137 IE 8 IE 135 IE 167 IE 4 IE 115 IE 227 IE 226 IE 225 IE 140 IE 136 P. djamor P. djamor P. djamor Wild isolate, Mexico Hybrid [IE 134 IE116] Japan, TFM-M-A-791 Japan, IFO Hong Kong (from Chang P1-27) Hybrid [IE 38 IE126] Cultivars, Guatemala (from R. de León) Czechoslovakia (from ICIDCA 166) Cultivars, Europe Czechoslovakia (from ICIDCA 184) Greece Deutschland (from Zadrazil P6) Cultivars, USA (from Fungi Perfecti) Hybrid [IE 4 IE115] Hybrid [IE 4 IE115] Hybrid [IE 4 IE136] Cultivars, USA (from Fungi Perfecti, C330) Czechoslovakia (from ICIDCA R)

4 1 MYCOLOGIA um, 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, 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. s 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. 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. 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). RESULTS When added to Cristomalt agar, Lysing Enzymes affected the mycelial radial growth of A. bisporus, L. edodes and (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. 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. 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 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

5 SAVOIE AND MATA: MUSHROOM ADAPTATION TO TRICHODERMA 195 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 Enzymes Agaricus bisporus Lentinula edodes Pleurotus ostreatus 5d 25.0 (2.34)* B 38.7 (0.77) C 45.9 (1.42) C YMEA 9d 33.5 (3.31) A 56.1 (1.28) B 75.8 (2.77) A 5d 19.6 (2.47) C 35.5 (0.74) D 40.9 (1.98) D YMEA Lysing 9d 24.6 (2.23) B 61.8 (0.97) A 66.9 (1.80) B * Mean and (standard deviation) from 20 replicates. The means associated to a same letter on a line are not significantly different (P 0.05), according to Fischer s LSD test. 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 1 (compounds with molecular weights higher than 10 Da), whereas no significant effect on biomass production was observed in 2. In 3 equivalent to 1 where enzymes were inactivated by heat treatment higher biomass was observed (TABLE V). wise, both Lysing Enzymes and 1 dramatically increased extracellular laccase activity, with a significantly higher effect for 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 2 and 3, but the difference was not significant with 1 (TABLE VII). Biomass production of T. aggressivum in assays was not different in Cristomalt, Cristomalt supplemented with Lysing Enzymes and Cristomalt supplemented with 1, whereas significantly higher biomass was produced with 2 or 3 (TABLE VII). 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 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 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 Mean biomass (mg 50 ml 1 ) Pairwise comparison probabilities in LSD Test Lysing Enzymes Lysing Enzymes

6 196 MYCOLOGIA 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 Laccase activity (nmol mg mycelium 1 ) Pairwise comparison probabilities in LSD Test Lysing Enzymes Lysing Enzymes 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. DISCUSSION 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 19, Score et al 1997). Lentinula edodes was shown to resist Trichoderma spp. attack by producing (1) large amounts of laccases (p-phenol oxidase, EC ) 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, -1,3- and -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 com- 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 With pre-culture of L. edodes Lysing Enzymes Biomass (mg 50 ml 1 ) Without pre-culture of L. edodes Pairwise comparison probabilities in LSD Test Lysing Enzymes

7 SAVOIE AND MATA: MUSHROOM ADAPTATION TO TRICHODERMA 197 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 Fungi Culture media Number of isolates A. bisporus 29 without L.E. First culture with L.E. Second culture with L.E. L. edodes 29 without L.E. First culture with L.E. Second culture with L.E. Pleurotus spp. 18 without L.E. First culture with L.E. Second culture with L.E. Mean growth rate (mm d 1 ) 3.3 E* 1.6 F 1.2 F 7.7 C 6.5 D 7.1 CD 12.6 A 10.1 B 11.6 AB 95% confidence interval [3.06; 3.57] [1.17; 1.96] [0.80; 1.51] [7.25; 8.19] [6.06; 6.96] [6.63; 7.54] [11.49; 13.74] [9.30; 10.96] [10.58; 12.61] * The means associated to a same letter on a line are not significantly different (P 0.05) according to Fischer s LSD test. pared 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 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

8 198 MYCOLOGIA 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. 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. ACKNOWLEDGMENTS This work was supported in part by Conseil Régional d Aquitaine Grant 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. LITERATURE CITED Ait-Lahsen H, Soler A, Rey M, De La Cruz J, Monte E, Llobell A An antifungal Exo- -1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum. Appl Environ Microbiol 67: Callac P, Billette C, Imbernon M, Kerrigan R A new

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