Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002UAEM. ISBN 968-878-105-3

GREEN MOLD DISEASE:ADAPTATION OF TRICHODERMA HARZIANUM Th2 TO MUSHROOM COMPOST

M. L. Largeteau-Mamoun1, G. Mata2 and J.M. Savoie1.1 UR430 Recherche sur les Champignons, INRA, BP81,

F-33883 Villenave d’Ornon Cedex, France ;2 Instituto de Ecologia, Departamento Hongos, Apdo. Postal 63,

Xalapa 91000, Veracruz, Mexico.<mamoun@bordeaux.inra.fr>

ABSTRACT

Green mold disease responsible for severe losses in Agaricus bisporus cultures emerged in Northern Ireland in 1985 and rapidly spread over England and the Netherlands. French mushroom farms were spared during a decade until the disease dramatically increased by the end of 1997. Molecular typing revealed Trichoderma harzianum Th2 was the pathogen in all European countries including France. Other Trichoderma species, such as T. longibrachiatum were responsible for minor damage. Differences in ability to colonize compost were observed between Th2 and non-Th2 isolates. The Th2 mycelium developed in unspawned commercial compost but non-Th2 showed only a few millimetres growth from the spore. Th2 germinated in spawned compost but non-Th2 did not. All Trichoderma spp grew intensively in sterilised compost and exhibited similar exocellular enzyme activities, revealing that adaptation of Th2 to compost is not nutrition related. Bacteria isolated from unspawned and spawned compost were screened for an inhibitory effect against mycelial growth of various Trichoderma spp Non-Th2 isolates were affected by a larger number of bacterial isolates than were Th2. When confronting A. bisporus, the latter did not induce a specific pigmented barrier acting as a resistance mechanism, as observed with other white-rot causing basidiomycetes. Adaptation of Th2 to compost, and antagonism towards A. bisporus resulted from better competitiveness against the compost microbial population. The recent outbreak of the disease in France may be explained by a delay in the use of short and aerated composting procedures compared to other European countries. The former procedures allowed the development of a more diversified microbial population, and consequently a larger number of potentially inhibitory organisms.

INTRODUCTION

Green mold disease of the button mushroom A. bisporus (Lange) Imbach, caused by Trichoderma harzianum specific biotypes Th2 and Th4, is responsible for important economic losses. The two aggressive biotypes were first found in the late 1980’s and do not show similarity with T. harzianum strains from culture collections (Muthumeenakshi et al. 1994, Ospina-Giraldo et al. 1998,1999). Less aggressive Trichoderma spp are responsible for occasional damage. Th4, the causal agent of green mold in America, is genetically distinct but closely related to Th2, the European pathogen (Muthumeenakshi and Mills 1995, Castle et al. 1998, Muthumeenakshi et al. 1998, Ospina-Giraldo et al. 1998). The disease appears in the mushroom cultivation substrate or in the casing layer as surfaces covered with Trichoderma spores. Emerging mushrooms are severely spotted, often distorted (Seaby 1989), making them unmarketable. In severe outbreaks no mushrooms are produced (Morris et al. 1995). Green mold disease appeared in Northern Ireland in 1985, arrived in Great Britain in 1987 (Seaby 1987 and 1989), and in the Netherlands in 1994 (Geels 1997). Four years elapsed before it was observed in the north of France by the end of 1997

179

(Védie and Olivier 1998, Mamoun et al. 2000b). Then, it was detected in Spain in 1998 (Hermosa et al. 1999). Short, aerated composting which restricts the variability of the microbial population in compost compared to previous procedures was used in France later than in other European countries. The objectives of this study were (i) to assess to what extend Th2 is responsible for the damage observed on French mushroom farms, (ii) to compare adaptation of Th2 and non-Th2 isolates to commercial compost in their development abilities and competition with bacteria isolated from this cultivation substrate, in order to understand how Th2 establishes and dramatically competes with A. bisporus.

MATERIALS AND METHODS

Trichoderma spp isolates

Trichoderma spp were collected at French mushroom farms from substrates showing green mold symptoms. Twenty-three isolates were from A. bisporus compost, six isolates from Lentinula edodes substrate, one from a cave wall (Table 1). The Th2 isolate T32, the Th1 isolates T20 and T41, and the Th3 isolates T18 and T43 (courtesy of Dr H. Grogan and Dr P. Mills, HRI, UK) were used as reference strains.

Table 1. Origin and genetic classification of the Trichoderma spp isolates used in this study.Isolate code Date Geographic

origin Substrate/other Compost yard

Symptoms at sampling Species/biotype

CE1 1995 Southwest L. edodes substrate * * T. longibrachiatumB Sept. 1997 North A. bisporus compost I Severe Th235 Sept. 1997 North A. bisporus compost I Minor T. longibrachiatumZ Sept. 1997 North A. bisporus compost II Severe Th2L Oct. 1997 Loire Valley A. bisporus compost V Minor undeterminedM Oct. 1997 North A. bisporus compost I Severe Th2V Oct. 1997 North A. bisporus compost III Severe Th2A Janv. 1998 Loire Valley A. bisporus compost V Minor undeterminedX1 Feb. 1998 Loire Valley A. bisporus compost V Medium Group aR June 1998 Loire Valley A. bisporus compost VII Severe Group aT June 1998 Loire Valley A. bisporus compost V Severe Group aC Feb. 1999 North Cave wall VIII * undeterminedZN Sept. 1999 North A. bisporus compost II Severe Th2RM Oct. 1999 Loire Valley A. bisporus compost IV Severe Th2S1 Dec. 1999 Southwest A. bisporus compost VI Severe Th2KA Feb. 2000 Brittany L. edodes substrate(1)a * * Th3KB Feb. 2000 Brittany L. edodes substrate (1) * * Group bP Feb. 2000 Brittany L. edodes substrate (1) * * Group bD March 2000 Loire Valley A. bisporus compost V Severe Th2G1 March 2000 Brittany L. edodes substrate (2) * * Group bG3 March 2000 Brittany L. edodes substrate (3) * * undetermined1021 Avr. 2000 Loire Valley A. bisporus compost V Severe Th21023 Avr. 2000 Loire Valley A. bisporus compost V Severe Th2LU May 2000 Loire Valley A. bisporus compost X Severe Th2MU May 2000 Loire Valley A. bisporus compost V Severe Th2PR May 2000 Loire Valley A. bisporus compost IV Severe Th2GM June 2000 Southwest A. bisporus compost VI Severe Th2509 June 2000 Loire Valley A. bisporus compost V Severe Th21017 June 2000 Loire Valley A. bisporus compost V Severe Th2MB Aug. 2000 Loire Valley A. bisporus compost V Severe Th3MS Aug. 2000 Loire Valley A. bisporus compost V Severe Th2a (1) , (2) and (3) = different batches of substrate.

180

RAPD analysis

DNA was extracted using the Nucleon PhytoPure extraction kit RPN 8510 (Amersham Int. plc, England) according to the manufacturer’s instructions. Amplifications were performed in 25 µl reaction mixture containing 0.1 mM dNTPs, 1X DyNAzyme™ buffer (Finnzymes Oy, Finland), 0.8 U DyNAzyme™ II polymerase (Finnzymes Oy, Finland), 0.5 µM decamer primer, 5 ng DNA. The Crocodile III Thermal Cycler (Appligene, France) was programmed for 1 cycle of 6 min at 94°C, 35 cycles of 1 min at 93°C, 2 min at 36°C, 2 min at 72°C, with a final extension period of 6 min at 72°C. Amplified products were visualised on 1.2% agarose gels, the 1Kb DNA ladder (Life Technologies) was used as size marker. Primers OPA03, OPA11, OPA13, OPB10, OpE16, OpH19, OPH20 (Operon Technologies, CA, USA), UBC29 (University of British Columbia, Vancouver, Canada) were screened.

Effect of spawning on Trichoderma spp development in mushroom compost

Five holes were made with a thin needle in the cover of petri dishes to allow aeration before they were filled with compost. Trichoderma spp inoculum consisted of a sterile rye grain coated with spores, as described by Romaine et al. (1998). A. bisporus was supplied as two spawn grains. Mycelial growth and sporulation of Trichoderma spp B, V, Z, 35 and F were observed in unspawned compost (one contaminated grain at the center of the dish) and in spawned compost (one contaminated grain and two spawn grains, each 4 cm from the others) (see Table 1). Plates were incubated for 23 days at room temperature and 10h light / 24 h. Five petri dishes were prepared for each treatment and the experiment was repeated twice. Mycelial growth and sporulation areas were drawn on the cover dish under a binocular microscope, and measured using a graphic integrator and a computer program.

Effect of compost composition on Trichoderma spp development

Sporulation rates of Th2 isolate Z were compared in various batches of compost in the presence of four strains of A. bisporus: the commercial strains S608 (Somycel) and 2400 (Amycel), and two wild strains (W1 and W2) from the INRA-CTC collection. Plastic boxes were filled with 200 g of spawned commercial compost inoculated with a single sterile rye grain coated with Trichoderma spp spores. After 23 days of incubation, Trichoderma spp sporulation was rated on a scale of 0 to 4, as described by Mamoun et al. (2000b).

Interaction on agar medium

Five basidiomycetes: A. bisporus U1 (Sylvan Spawn), L. edodes S610 (France Mycelium), Lepista nuda Ferland (Sylvan Spawn), Pleurotus ostreatus (INRA, PoJMO97),and P. eryngii (INRA, PeS85) were challenged by the isolates T. harzianum Th2 B and T. longibrachiatum CE1. Each basidiomycete was cultivated on YMEA medium 6 days at 24°C and then inoculated with Trichoderma spp at a distance of 4 cm (Savoie et al. 2001b). Five petri dishes for each basidiomycete/Trichoderma spp combination were observed daily for evidence of contact area and the growth of Trichoderma spp on the basidiomycete mycelium.

Effect of bacteria isolated from compost on the growth of Trichoderma spp

Bacterial colonies were isolated from commercial compost before spawning (BS) and on day 3 after spawning (S+3), and purified. Twenty-seven isolates were confronted with Trichoderma spp on compost extract agar. Bacteria were placed 5 cm from the Trichoderma spp inoculum. After seven days of incubation at 23°C, the surface covered with mycelium was compared to the control

181

(without bacteria) by analysis of variance (SAS system, SAS Institute Inc., Cary, NC, USA). The effect of the bacteria on A. bisporus strain S608 was compared using a similar procedure.Nine randomly chosen colonies were screened for diversity with the API 20NE system (BioMérieux, France) according to the manufacturer’s protocol.

RESULTS

RAPD analysis

Seventeen isolates showed a Th2 pattern when compared to the reference isolate T32. All were from A. bisporus cultures and responsible for severe damage at the sampling date. A unique profile was obtained with six primers. Isolates ZN and LU exhibited polymorphism with primers OPH19 (Figure 1) and OPE16, respectively. Eleven of the twelve isolates collected from mushroom compost in 2000 were Th2, and the last one was Th3. The Th1 biotype was found neither in compost nor in L. edodes substrate. Th3 and T. longibrachiatum were detected in each substrate (Figure 2). Ten isolates were not identified. Among these, groups a and b showed homogeneous RAPD patterns with all primers. Th2 and non-Th2 gave green mold symptoms at the same period on the same farm: T. longibrachiatum isolate 35 and Th2 isolate B, Th3 isolate MB and Th2 isolate MS in mushroom compost, Th3 isolate KA and non-Th2 group b in L. edodes substrate (Figures 1 and 2, Table 1).

Figure 1. RAPD patterns of T32 and 17 French Th2 isolates with primer OPH19.NC = negative control, without DNA. The arrow shows the additional band of isolate ZN.

Figure 2. RAPD analyses of non-Th2 isolates with primer OPB10.NC = negative control, without DNA, T.long = T. longibrachiatum, * = collected from L. edodes substrate.

B D GM

LU M MS

MU

PR RM

S1 T32

V Z ZN 509

1017

1021

1023

NC

MS

MB

CE1

35 T40

T41

T18

T43

R T X1

L A C P G1

KB

KA

G3

NC

Th3 T.long Th1 Th3 Group a Group b Th3

* * * * * *

182

Effect of spawning on Trichoderma spp development in mushroom compost

Th2 mycelium flourished in compost unspawned or spawned with A. bisporus, but the mycelium of the button mushroom is required for intensive sporulation. The non-Th2 isolates screened failed to grow and sporulate significantly in both composts (Figure 3).

Effect of compost composition on Trichoderma spp development

Important variations in the sporulation of Th2 isolate Z were observed in the presence of the same A. bisporus strain depending on the batch of compost and on the replicate of the same compost (Figure 4). The mean rate of sporulation depended on the A. bisporus strain, but Trichoderma spp sometimes sporulated intensively in the presence of strains considered to be poorly susceptible such as W2 or failed to sporulate in the presence of a susceptible strain such as S608. Similar results were obtained with other Th2 isolates and other A. bisporus strains (data not shown).

Figure 3. Comparison of mycelial growth and sporulation ofTh2 and non-Th2 isolates in spawned and unspawned compost.

Figure 4. Effect of compost composition on sporulation (classes 0-4) of Th2 isolate Z.For each Agaricus bisporus strain (S608, W1, 2400, W2), data represent at least four composts,each with six replicate boxes. Arrows indicate the mean class for sporulation rate.

183

Interaction on agar medium

When confronted either with T. longibrachiatum isolate CE1 or Th2 isolate B, L. edodes formed a brown line which acted as a barrier against the pathogen. P. ostreatus and P. eryngii showed this defensive reaction with CE1, but they did not produce the pigmented barrier in the presence of the Th2 isolate which covered the mycelium of the basidiomycetes. A. bisporus was susceptible to both species of Trichoderma which grew intensively on its mycelial colony and no brown line was observed (Table 2).

Table 2. Brown line formation and overgrowth of Trichoderma spp on the mycelium offour basidiomycetes after confrontation on agar medium.

Basidiomycetes T. longibrachiatum CE1 T. harzianum Th2 BBrown line Overgrowth Brown line Overgrowth

A. bisporus - + - +L. nuda - + - +L. edodes + - + -P. ostreatus + - - +P. eryngii + - - +

Effect of bacteria isolated from compost on the growth of Trichoderma spp

The formula obtained with the API 20NE system for each of the bacterial colonies was unique (Table 3).

Table 3. Formula obtained with the API 20NE system for the nine randomly chosen bacterial colonies.

Formula Numbers of bacteriaIsolated at BS Isolated at S+3

1457744 1 01567741 1 01447025 1 01467745 1 01567740 1 00477745 0 11457345 0 11457740 0 11453307 0 1

The mycelial growth of the Th2 isolates B and Z was affected by a smaller number of bacterial colonies than was the weakly aggressive T. longibrachiatum isolate 35. The non-Th2 isolate X1, responsible for medium-to-severe damage, resisted the majority of the bacterial isolates (Table 4). No bacterium was found to increase Trichoderma spp growth. Bacteria affecting Th2 growth had no effect on A. bisporus mycelial growth. Bacteria affecting Th2 growth had no effect on A. bisporus mycelial growth.

184

Table 4. Distribution of the 27 bacterial isolates according to their effect on the mycelium growthof Trichoderma spp on day 7 after confrontation.

Trichoderma spp

Symptoms Numbers of bacterial colonies / effect

Isolate at sampling Stable inhibition Unstable inhibition * No effectB Severe 3 2 22Z Severe 4 5 18X1 Medium to severe 2 0 2535 Minor 10 3 13

* Inhibition significant (P<0.05) on day 3 and not significant on day 7 after confrontation was considered as unstable.

DISCUSSION

Trichoderma harzianum biotype Th2 was responsible for the outbreaks of green mould disease in the North of France by the end of 1997 (Mamoun et al. 2000b). In two years, Th2 had spread throughout France, and represented almost all of the isolates collected from A. bisporus cultures in 2000. Only one isolate was identified as Th3, Th1 was not detected. At the same period, on the same mushroom farm, two biotypes or two species can grow together either in Agaricus compost or in L. edodes substrate.

Th2 develops and sporulates in sterilized compost (Savoie et al. 2001a). In commercial compost, the mycelium of A. bisporus is essential for Th2 to sporulate intensively. As soon as sporulation occurs, mycelial growth of A. bisporus is dramatically reduced and typical green mould rapidly develops (Mamoun et al. 2000a). Some enzymes produced by various Trichoderma species are supposed to be connected either to their saprophytic lifestyle or to their direct action against other fungi (Sivan and Harman 1991, Silveira et al. 1997, Antal et al. 2000, Thrane et al. 2000). Th2 has been shown to be genetically different from other T. harzianum biotypes (Ospina-Giraldo et al. 1999). Therefore, differences in enzyme secretion were expected. When 17 secreted enzymes were screened (enzymes able to depolymerise organic components in compost and in A. bisporus cell walls into absorbable forms), there was no specific difference between Th2 and non-Th2 isolates grown in sterilized compost (Savoie et al. 2001a). Th2 isolates have no specific ability to produce enzymes necessary for compost degradation. The Th2 isolates screened resisted a larger number of bacteria isolated from mushroom compost than did a non-Th2 isolate inducing minor symptoms. Results obtained with API systems indicated that the bacterial isolates screened for their effect on Trichoderma spp represented a diversified sample. This inhibitory effect of bacteria is thus an important parameter influencing the competitive saprophytic ability of Trichoderma spp in mushroom compost. Differences in the rate of Th2 colonisation were observed in various batches of compost, as was the extension of the Th2 mycelium. Th2 appears as round circles in sterile compost, but as irregular shapes in commercial compost, as Mamoun et al. (2000b) also observed.The ability of the Th2 biotype to colonize mushroom compost before contact with A. bisporus mycelium therefore depends both on resource capture and the capacity to resist microbial antagonists. Lyses of A. bisporus hyphae by Th2 may be responsible for the aggressive nature of this biotype of T. harzianum when nutrients become limited in the mushroom compost (Mumpuni et al. 1997). A. bisporus does not produce a defense mechanism as observed in the other cultivated mushrooms screened, which makes it particularly susceptible to Th2 and other Trichoderma spp.

The recent outbreak of the disease in France may be explained by the fact that short, aerated composting procedures continue to be used there more than in other European countries. This

185

procedure permits a more diversified microbial population to develop, and consequently selects a larger number of potentially inhibitory organisms.

ACKNOWLEDGEMENTS

We wish to thank mushroom growers and R. Vedie, A. Rodier, T. Rousseau (Mushroom Technical Center, CTC, Distre, France) for Trichoderma spp samples; and T. Gibard and N. Minvielle for technical assistance. We are grateful to K. Mayo-Candresse for reviews of the English version of the manuscript. This project was partly funded by the Conseil Régional d’Aquitaine (France) and the ECOS-Nord Committee (Mexico).

REFERENCES

Antal, Z., L. Manczinger, G. Szakacs, R.P. Tengerdy and L. Ferenczy. 2000. Colony growth, in vitro antagonism and secretion of extracellular enzymes in cold-tolerant strains of Trichoderma species. Mycol. Res. 104: 545-549.

Castle A., D. Speranzini, N. Rghei,G. Alm, D. Rinker and J. Bisset. 1998. Morphological and molecular identification of Trichoderma isolates on North American mushroom farms. Appl. Env. Microbiol. 64: 133-137.

Geels, F.P. 1997. Ronde tafel-bijeenkomst over Trichoderma. Champignoncultuur 41: 13.Hermosa, M.R., I. Grondona and E. Monte. 1999. Isolation of Trichoderma harzianum Th2 from commercial

mushroom compost in Spain. Plant Disease, June 1999: 591.Mamoun, M.L., J.-M. Savoie and J.-M. Olivier. 2000a. Interactions between the pathogen Trichoderma

harzianum Th2 and Agaricusus bisporus in mushroom compost. Mycologia 92: 233-240.Mamoun, M.L., R. Iapicco, J.-M. Savoie and J.-M. Olivier. 2000b. Green mould disease in France :

Trichoderma harzianum Th2 and other species causing damages on mushroom farms. Mush. Sc.15: 625-632.

Mumpuni, A., H.S.S. Sharma and A.E. Brown. 1997. A possible role of lytic enzymes produced by Trichoderma harzianum biotypes during interaction with Agaricus bisporus. In: R. D. Rai, B. L. Dhar and R. N. Verma (eds) Advances in Mushroom Biology and Products. MSI Solan, India.225-229.

Muthumeenakshi S., P.R. Mills, A.E. Brown and D.A. Seaby. 1994. Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles. Microbiology 140: 769-777.

Muthumeenakshi S. and P.R. Mills. 1995. Detection and differentiation of fungal pathogens of Agaricus bisporus. Mush. Sc. 14: 603-610.

Muthumeenakshi S., A.E. Brown and P.R. Mills. 1998. Genetic comparison of the aggressive weed mold strains of Trichoderma harzianum from mushroom composts in North America and the British Isles. Mycol. Res. 102: 385-390.

Ospina-Giraldo, M.D., D.J. Royse, M.R. Thon, X. Chen and C.P. Romaine. 1998 Phylogenetic relationships of Trichoderma harzianum causing mushroom green mold in Europe and North America to other species of Trichoderma from world-wide sources. Mycologia 90: 76-81.

Ospina-Giraldo, M.D., D.J. Royse, X. Chen and C.P. Romaine. 1999. Molecular phylogenetic analyses of biological control strains of Trichoderma harzianum and other biotypes of Trichoderma spp associated with mushroom green mold. Phytopathology 89: 308-313.

Romaine C.P., D.J. Royse, P.J. Wuest and D.M. Beyer. 1998. Mushroom green mold: cause, edaphic factors and control. Mushroom News 46: 12-17.

Savoie J.-M., R. Iapicco and M.L. Largeteau-Mamoun. 2001a. Factors influencing the competitive saprophytic ability of Trichoderma harzianum Th2 in mushroom (Agaricus bisporus) compost. Mycol. Res. (in press).

Savoie, J.-M., G. Mata and M. Mamoun. 2001b. Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2. Mycologia 93: 243-248.

Seaby D.A. 1987. Infection of mushroom compost by Trichoderma species. Mushroom J. 179: 355-361.

186

Seaby D.A. 1989. Further observations on Trichoderma. Mushroom J. 197: 147-151.Silveira, F.Q.P., I.S. Melo and E.X.F. Filho. 1997. Carbohydrate-hydrolysing enzyme activity production by

solid-state cultures of Trichoderma harzianum strains. Revista de Microbiologia 25: 152-156.Sivan, A. and G.E. Harman. 1991. Improved rhizosphere competence in a protoplast fusion progeny of

Trichoderma harzianum. Journal of General Microbiology 137: 23-29.Thrane, C., D. Funck Jensen and A. Tronsmo. 2000. Substrate colonization, strain competition, enzyme

production in vitro, and biocontrol of Pythium ultimum by Trichoderma spp isolates P1 and T3. Eur. J. Plant Pathol. 106: 215-225.

Védie R. and J.-M. Olivier. 1998. La moisissure verte Trichoderma: un problème à prendre très au sérieux. Bull. FNSACC: 665-669.

187