A challenge to mushroom growers: the green mould disease of cultivated

champignons

L. Kredics1, L. García Jimenez

1, S. Naeimi

2, D. Czifra

1, P. Urbán

1, L. Manczinger

1, C. Vágvölgyi

1, and

L. Hatvani1

1 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52. H-6726 Szeged, Hungary

2 Department of Biological Control Research, Iranian Research Institute of Plant Protection, Tehran, Iran

The aim of this minireview is to give an overview of the literature about the green mould disease of cultivated champignons (Agaricus bisporus) caused by Trichoderma species. The first significant green mould epidemic appeared in Northern Ireland in 1985, which was quickly followed by subsequent outbreaks in several countries. The symptoms of green mould appear as large patches of compost turning green rapidly. Epidemic outbreaks are due to two varieties of the species T. aggressivum. This species competes efficiently for space and nutrients, produces extracellular enzymes, toxic secondary metabolites and volatile organic compounds, which results in drastical crop losses. The natural habitat of T. aggressivum is still unknown. Possible routes of infection include the air, vehicles, contaminated clothes and animal vectors. A primer pair for the specific identification of T. aggressivum is available for the fast and cheap monitoring of the causal agent. Possible management strategies include the application of disinfectants, pasteurization, adjustment of casing pH, chemical treatments, biological control by antagonistic bacteria as well as the cultivation of resistant Agaricus varieties.

Keywords champignon; Agaricus bisporus; Trichoderma aggressivum; green mould disease, mushroom production

1. Introduction

Trichoderma species are asexual, soil-inhabiting filamentous fungi with teleomorphs belonging to the genus Hypocrea (Ascomycota, Pyrenomycetes, Hypocreales, Hypocreaceae). Besides the industrial importance of the genus [1], certain Trichoderma species are well known to have the ability of antagonizing a series of plant pathogenic fungi [2]. Proposed mechanisms of antagonism include mycoparasitism by the action of cell-wall degrading enzymes, antibiosis by the production of antibiotics, competition for space and nutrients through rhizosphere competence, facilitation of seed germination and growth of the plants via releasing important minerals and trace elements from soil and induction of the defense responses in plants [3–5]. During the last decades, representatives of the genus have been reported to be harmful as emerging opportunistic pathogens of humans [6, 7] and as the causative agents of green mould disease, a disorder that results in substantial losses in the production of cultivated mushrooms including champignon (Agaricus bisporus), shiitake (Lentinula edodes) and oyster mushroom (Pleurotus ostreatus).

2. Green mould disease of champignons: a historical overview

World-wide mushroom cultivation is dominated by the production of Agaricus bisporus (champignon), which is followed by Lentinula edodes (shiitake) and Pleurotus ostreatus (oyster mushroom) [8]. Infections in mushroom cultivation due to members of the genus Trichoderma have come to be known as weed mould, or more commonly as the “green mould disease”. For A. bisporus, the association of Trichoderma species with the respective compost has been known for a long time to limit commercial production [9]. Historically, Trichoderma viride and Trichoderma koningii were reported to cause losses in cultivation of champignons episodically [9]. Sinden [10] considered the genus Trichoderma as species competing with the mushroom or indicator of poor compost, associating their presence to situations with acidic pH or soluble sugar residues. Until the 80’s, mushroom green mould was considered as a minor problem only, associated mainly with the low quality of the compost or poor hygiene, which could be managed effectively by modifying the composting process, improving sanitation or chemical intervention [11]. This point of view changed basically after the first green mould epidemics in the British Isles during 1985-1986 and in late 1990 and 1991 which caused losses of about £ 3-4 million [12–20]. There were also serious losses in the Netherlands in 1994 [21]. In the early 1990s, a similar disease appeared in mushroom crops in North America (Alberta, Ontario, British Columbia, and Pennsylvania) causing losses of more than $ 30 million [22–28]. In France, the disease was detected in 1997 [29, 30]. In Spain, the first observations of Trichoderma strains, much more aggressive than the ones known previously, were made by plant breeders in La Rioja during the winter of 1996-1997 [31,32]. It was subsequently observed in culture vessels from the same town and at the end of the campaign the disease spread to the neighboring towns. During the past decade, the Trichoderma green mould disease of A. bisporus also appeared in Hungary [33], Poland [34–36],

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Croatia (Hatvani et al. unpublished), Mexico [37] and Australia [38]. The escalation of the green mould problem in Agaricus cultivation evoked extensive research efforts to identify and study the causal agents.

3. The causal agents of Agaricus green mould

Although a number of Trichoderma spp. (e.g. T. koningii, T. hamatum, T. longibrachiatum, T. citrinoviride, T. crassum, T. spirale [25]) have been isolated from mushroom compost, aggressive colonization resulting in epidemic outbreaks were attributed originally to T. harzianum only [13, 16, 17]. Compost isolates from the British Isles identified as T. harzianum have been differentiated into three biological forms [13, 16]. Biotypes Th1, Th2 and Th3 [16] were found to differ in their growth rates, sporulation patterns and aggressiveness in compost colonization, with Th2 being the aggressive form responsible for green mould epidemics based on inoculation experiments [12, 16, 17, 39]. (The term “aggressiveness” refers in this review to the reduction in A. bisporus yield). The biotypes could also be differentiated by colony appearance, as well as micromorphological features including phialides and phialospores [18]. Biotype Th1 is commonly found in compost raw materials and yards but rarely found in bags of pasteurized compost [16]. It grows quickly (1mm/h) at 27 °C and sporulation occurs in two days after exposure to light, creating a lot of aerial mycelium. Sporulated tissue acquires green colour similar to spinach. The crop has malt smell [31]. Th2 is predominantly present in affected compost but rarely in compost raw material [14]. It grows quickly (1mm/h) at 27 °C producing a cottony layer of aerial mycelium. Sporulation does not occur until at least four days and then arises in the central area in green concentric bands. Th3 is found in raw materials and yards but seldom in affected bags, trays or shelves except in cases where dust from ingredients could have blown onto the pasteurized compost [18]. It grows at a rate of 0.5-1 mm/h. The colonies have a radial aspect and the culture smells like coconut. This initial grouping was later confirmed during the investigation of 81 Trichoderma strains isolated from mushroom compost by a series of molecular techniques, including restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) with six primers and the sequence analysis of the internal transcribed spacer 1 (ITS1) region [38]. Genetic uniformity was found in the case of Th2 [40], which supports the hypothesis that the green mould agent throughout the British Isles may have originated from a single source, possibly in Northern Ireland [14], which was derived from a mutant particularly suited for growth on mushroom compost [16]. However, minor variation in mitochondrial DNA (mtDNA) can distinguish Irish strains from those in Great Britain [40]. This genetic variation may be due to a large number of mutational events after the first mutation that allowed the initial colonization of the compost. The abovementioned molecular techniques were used later for the molecular characterization of Trichoderma strains isolated from North American mushroom farms [25, 41–43]. The aggressive group Th2 in British Isles was found to be different from the one observed in North America (group Th4). Strains of biotype Th4 appeared to be genetically uniform, suggesting that Th4 strains may be originated from a single source. The difference in the ITS1 sequence was 5 base pairs between biotypes Th2 and Th4, and the ITS1 sequence analysis revealed that these two biotypes are phylogenetically closely related to T. harzianum group Th1 [42]. The growth rate of Th4 is 0.8 mm/h, the colonies produce aerial mycelium and have wavy edges. Sporulation occurs in bands [18]. Based on these results it was concluded that the green mould disease was not caused by a single strain, creating the alternative hypothesis that aggressive forms emerged from at least two independent sources (in the British Isles and in North America) by the adaptation of existing populations to environmental conditions of mushroom production. This hypothesis explains the differences between the North American and the Irish and British isolates. As T. harzianum is a species often used for biological control of fungal plant pathogens, concerns have emerged regarding the possible involvement of biocontrol strains in the development of mushroom green mould. However, molecular phylogenetic studies based on RAPD analysis as well as the sequence analysis of the ITS1-5.8S rDNA-ITS2 region revealed that although biocontrol and green mould isolates were closely related, they could be clearly distinguished from each other [27, 44, 45], suggesting that Th2 and Th4 have evolved from a recent common ancestor

for both biocontrol and green mould-related biotypes. Phylogenetic analysis of the β-tubulin gene supported the above findings [46], furthermore, pathogenicity trials also indicated that commercial biocontrol T. harzianum strains and related ones from the Th1 biotype were not pathogenic on A. bisporus, in contrast to Th4 isolates [47, 48]. Based on the molecular differences between the biotypes Th1-3, Muthumeenakshi et al. [40] already suggested that they might represent three different species. Molecular evidences indicated later that biotype Th3 was actually T. atroviride [25, 26], while Th1 was recognized as T. harzianum sensu stricto [49]. These two species were found to be the most common Trichoderma species in the Australian mushroom industry [38]. More recently, the two aggressive biotypes, Th2 and Th4 were redescribed on the basis of morphological characteristics and the phylogenetic analyses of ITS1 and the translation elongation factor 1-alpha (tef1) gene as T. aggressivum f. europaeum and T. aggressivum f. aggressivum, respectively [50]. T. aggressivum f. europaeum is responsible for the green mould problems in Europe, while T. aggressivum f. aggressivum is known as a pathogen of cultivated A. bisporus in Canada, USA and Mexico. A project undertook a survey of Trichoderma species present in UK mushroom production on 15 farms over a 6-month period, commencing in December 2007, but T. aggressivum f. aggressivum was not found [51]. It is not known whether the North American biotype poses an additional risk to UK mushroom production [52]. The introduction of the North

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American strain would increase the genetic diversity of the T. aggressivum species complex in Europe, which could present new, unforeseen problems to European mushroom growers. Apparently this has already happened in the case of Australia, where T. aggressivum f. aggressivum has been found on one farm, where it could also be associated with yield losses [53]. The biotypes formerly known as Th2 and Th4 will be further referred to in this review as Ta2 and Ta4, respectively. Although T. aggressivum f. europaeum (Ta2) and T. aggressivum f. aggressivum (Ta4) have subtly different growth rates at 25ºC on synthetic low nutrient agar (SNA) and statistically significant differences in micromorphology, they are effectively indistinguishable based on morphology only [50].

4. Pathogenesis and epidemiology

4.1 Symptoms of Agaricus green mould disease

Pathogenic green moulds may colonize the substrate or grow on the surface of the emerging mushrooms. No symptoms appear in the bags until 10-35 days following the apparently normal spawn run of A. bisporus. Trichoderma spp. produce whitish mycelia indistinguishable from those of the mushrooms during spawn run, therefore it is difficult to recognize the infection at this stage [54]. Subsequently, large patches of compost turn green rapidly as spore production begins on the Trichoderma mycelia which had run through the compost with Agaricus [18, 55] (Fig. 1). Morris et al. [14, 15] described the symptoms of green mould disease as the presence of green fungal sporulation in the mushroom compost or casing layer between 2-5 weeks of the production cycle in the mushroom growing unit. The crop loss is proportional to the area infected, where generally no mushrooms are produced in contaminated bags in the case of serious outbreaks. Furthermore, even if mushrooms do appear, they are unsaleable as they often become severely spotted, distorted [17] and infested with red pepper mites (Pygmephorus mesembrinae), which feed on the Trichoderma and gather on the mushrooms [14, 15].

4.2 Interactions between Trichoderma and Agaricus in the compost

Parameters of mushroom cultivation, such as the sources of carbon and nitrogen, high relative humidity, elevated temperature, a fluctuation of these factors and the absence of light during spawn run are ideal environmental conditions for Trichoderma species as well, which can easily result in a contamination. The speed and magnitude of compost colonization by Trichoderma is influenced by key compost characteristics like moisture, ash content and the degree of fermentation [56]. Compost parameters with a positive influence on Agaricus are also favourable for the colonization of Trichoderma. Among preferred conditions, moulds like Trichoderma exhibit fast growth, therefore they can compete for space and nutrients more effectively than the mushrooms, furthermore, they are able to produce extracellular enzymes, toxic secondary metabolites as well as volatile organic compounds [57, 58], which can result in a drastical decrease in production or even entire crops can be wiped out.

Fig. 1 Symptoms of green mould caused by Trichoderma aggressivum f. europaeum on compost used for Agaricus cultivation.

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Compost contains a large amount of lignin and cellulose, and thus is a medium highly selective for basidiomycetes capable of utilizing these constituents as carbon sources through the activity of extracellular enzymes such as laccase [59]. Thus, compost-colonizing Trichoderma biotypes require the presence of A. bisporus mycelium in order to proliferate through the compost [16, 18]. Different opinions emerge from the literature about the development of T. aggressivum f. europaeum (Ta2) in compost. Seaby [18] reported that Ta2 mycelium did not establish visibly in the absence of A. bisporus. Romaine et al. [60] also concluded that green mould grew hardly in compost without A. bisporus. Rinker [55] suggested that aggressive Trichoderma could grow in compost without A. bisporus, however it flourished only in newly inoculated compost. Based on the findings of further studies, Ta2 is able to colonize compost both inoculated and not inoculated with A. bisporus [14, 15, 29]. The results of Mamoun et al. [29] provided an explanation for the above listed, different findings: actually the mycelium of A. bisporus is required for the induction of intensive sporulation by Ta2, which has the ability to colonize compost also in the absence of A. bisporus, however, its very thin hyphae make it undetectable without a microscope. The ability of Ta2 to attain a large active mass of mycelium along with delayed sporulation may be the key to colonizing spawned compost and giving rise to the reduction of both champignon quality and yield. Sharma et al. [56] reported that the Ta2 isolates inoculated into the compost at spawning readily became established and spread rapidly in all directions. In contrast, T. harzianum (Th1) and T. atroviride (Th3) established slowly and grew only a short distance from the point of inoculation, thereby reducing the quality of mushrooms but not affecting the crop yield significantly. Mumpuni et al. [57] reported that biotypes Th1, Ta2, and Th3 produced volatile metabolites in vitro that had similar fungistatic effects on the growth of Agaricus bisporus. Higher levels of toxicity were produced by Th1 and Th3 (and not by Ta2, which might have been expected to produce the greatest toxic effect on A. bisporus). Moreover, Th1 and Th3 are more strongly inhibited by a compound(s) produced by A. bisporus than Ta2, and are usually confined to small localized areas in the compost from which A. bisporus is excluded [16]. These findings suggest mutual inhibition by A. bisporus and Th1 and Th3. The stimulation of Ta2 by a compound(s) produced by A. bisporus was also reported [57]. The authors hypothesized that this stimulation along with the relative tolerance of A. bisporus for toxins produced by Ta2 might allow the simultaneous growth of the two fungi in compost. This simultaneous growth of A. bisporus and Ta2 can be observed before the mycelium of Agaricus stimulates Ta2 sporulation. As soon as sporulation appears, the mycelial growth of A. bisporus is reduced dramatically and the typical green mould develops rapidly [29]. Krupke et al. [61] identified a metabolite, 3,4-dihydro-8-hydroxy-3-methylisocoumarin, produced by T. aggressivum f. aggressivum isolates in vitro, which was not produced by ‘non-aggressive’ Trichoderma isolates. This compound inhibits the growth and subsequent fruiting body formation of A. bisporus during the establishment of green mould disease, allowing Ta4 to proliferate and utilize nutrients liberated from compost constituents by A. bisporus extracellular enzymes. Ta4 needs extracellular enzymes produced by A. bisporus to break down the complex compost constituents into simpler carbon sources that can be absorbed and utilized [61]. Trichoderma species are able to produce a range of extracellular hydrolytic enzymes that degrade different polymers, which can thus be used as nutrient sources. In the case of compost inhabiting Trichoderma strains, extracellular β-1,3 glucanases have substrates in the cell wall of both A. bisporus and wheat straw (the main component of mushroom compost), while chitinases and proteases may facilitate saprotrophic growth on the rich fungal and bacterial compost microflora. No specific differences could be observed between Ta2 and non-aggressive strains in the activities of 17 extracellular enzymes tested [54, 62]. On the other hand, confrontations of 27 bacterial isolates with Trichoderma strains revealed in these studies that the aggressive Ta2 strains were affected by a lower number of bacterial isolates than the non-aggressive strains, suggesting that the better adaptation of Ta2 to mushroom compost is probably due to the ability to tolerate inhibitory effects of bacteria present in mushroom compost, and not an ability to degrade its components. This makes it possible that Ta2 colonizes certain areas of the compost before interacting directly with A. bisporus. In a second period the lysis of A. bisporus hyphae by Ta2 might be responsible for its aggressive nature against A. bisporus, when the nutrients in compost become limiting [57]. Williams et al. [58] examined the mycoparasitic and saprotrophic behavior of compost inhabiting Trichoderma isolates representing groups Th1 (T. harzianum), Th3 (T. atroviride), as well as Ta2 and Ta4 to establish a mechanism for the aggressiveness towards A. bisporus in infested compost. Mycoparasitic structures could be observed infrequently, suggesting that the antagonism of T. aggressivum to A. bisporus is not the result of mycoparasitism primarily. All examined Trichoderma groups secreted polymer-degrading extracellular enzymes that could attack the cell walls of A. bisporus and of wheat straw, and some of them, e.g. chymoelastase and trypsin-like proteases - produced by groups Ta2 and Ta4 only - were found to be linked to aggressiveness. This suggested that polymer-degrading extracellular enzymes were essential for both saprotrophic and parasitic growth. However, some Th3 (T. atroviride) isolates were able to colonize sterilized compost at levels similar to those of T. aggressivum and also to produce equivalent levels and ranges of polymer-degrading extracellular enzymes, suggesting that aggressiveness can not be explained by these factors only. The authors proposed that aggressiveness was dependent on extensive saprophytic colonization and presumably on associated competition, but competition and colonization could be prerequisites to the antagonism to A. bisporus, of which mycoparasitism could be just one of several components [58].

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Guthrie and Castle [63] determined the activities of intracellular and extracellular chitinases produced in dual cultures of Ta4 and A. bisporus over a 2 week period. The authors suggested that one of the 3 N-acetylglucosaminidases detected in A. bisporus (M: 96 kDa) might have a role in the resistance of commercial brown strains to green mould disease, while one of the 3 N-acetylglucosaminidases detected in Ta4 (M: 122 kDa) may be an important predictor of the antagonistic activity.

4.3 Epidemiology of Agaricus green mould disease

Regarding the possible sources of infection, T. aggressivum has not yet been isolated from the natural environment. In the case of the pathogens of the green mould disease of Pleurotus ostreatus, we have found recently that one of the pathogenic Trichoderma species, T. pleuroticola is present in the growing substrates and on the surface of the basidiomes of wild growing oyster mushrooms [64]. In order to examine whether the natural substrates and the surface of wild-growing Agaricus species are possible reservoirs of T. aggressivum, we isolated Trichoderma strains from three samples from the environment of wild-growing Agaricus species in Hungary (Kecskemét, Nagykőrös, Szeged; Czifra et al., unpublished). A total number of 65 Trichoderma isolates derived from Agaricus spp. were studied both by a PCR-based diagnostic test for T. aggressivum (see section 5. for details of the method) and by the sequence analysis of the ITS region and a gene fragment containing the 4th and 5th introns of the tef1 gene. Based on the sequences, 7 Trichoderma species were identified with the aid of the programs TrichOKEY 2.0 [65] and TrichoBLAST [66], both available online by the International Subcommission on Trichoderma and Hypocrea Taxonomy: T. atroviride, T. tomentosum, T. hamatum, T. harzianum, T. koningii, T. koningiopsis and T. virens. T. aggressivum was not found in the examined samples derived from the natural environment of wild Agaricus spp. (Czifra et al., unpublished), so we still have no data about its occurrence outside of mushroom cultivation. Initial expansion of the T. aggressivum f. europaeum epidemic in the British Isles might have occurred from one compost producer to another via mutual customers. Vehicles contain large amounts of dust, spores, mycelial debris, mosquitoes and mites, so their movement is one of the risk factors of spreading among different facilities. Furthermore, during the original epidemic, thousands of bags green from Trichoderma were dumped by growers or spread on the fields, which might have also contributed to the initial spreading of the disease [19]. Affected farms frequently experience persistent green mould problems and stringent sanitation procedures are required to minimize the possibility of infection of the freshly spawned compost with green mould [16, 67]. A further important question is the route of the infection. According to a producer’s record, levels of green mould contamination were 60% higher in weeks with dry, windy conditions during compost bagging, furthermore, peak heat tunnels most exposed to windborne contamination had the worst records, suggesting that airborne dust may provide a major contribution to contamination [19]. Seaby [19] performed detailed epidemiological investigations regarding the Trichoderma green mould caused by Ta2 in Northern Ireland and the Republic of Ireland, where the satellite method (compost packed in discrete 20 kg units) is applied for Agaricus production. The study involved both the investigation of samples taken from compost and mushroom producing facilities, and artificial infections experiments. No Ta2 was found in samples of lagoon water from production yards. Nearly all surfaces in affected mushroom houses and bagging areas (bagging machinery, floors in production units, hand rails, ladder rungs, spawn hoppers, tarpaulin road covers, trailers, wooden pallets), as well as rest-room tables, chairs and handles touched by mushroom pickers were found to be positive for Ta2. Clothing of workers (woollen jerseys, jackets, picker’s sleeves) examined by electrostatic or vacuum sampling also yielded Trichoderma species, most frequently Ta2. No Ta2 isolates could be recovered from peak-heated jars of contaminated compost, indicating that Ta2 did not survive peak heating. Isolations from cotton bags decreased to zero after 30 min of tumble drying at 60ºC. Potential biological vectors of the green mould disease were also evaluated: microscopic examination of red pepper mites revealed clusters of Trichoderma spores attached on their bodies, and Ta2 could be isolated from them. Red pepper mites appear to reproduce on all of the compost-related Trichoderma species, therefore they may not be indicators of which species of green mould is present [38]. Ta2 could also be isolated from sciarid mushroom flies, furthermore, when the cadaver of a mouse carrying mites was placed in contact with spawn, it became infected by green mould [19]. Artificial infection experiments revealed that an early inoculation at day 7 led to heavy Ta2 development, while a late inoculation at day 14 did not result in colonization [19]. Contaminated CACing (chopped compost added to casing to speed spawn run) did not result in Ta2 development, however, certain field cases suggested that Ta2 sporulation on the surface of bags might be associated with the addition of contaminated compost as CACing in the casing mix. When readily spawned compost bags were touched by hands artificially contaminated wit Ta2, all bags became heavily infected. The application of metals essential for Agaricus growth (Cu, Zn, Fe and Mn) was not found to reduce colonization by Ta2, while bacterial isolates from compost could suppress its growth [19]. Royse et al. [68] examined the spatial distribution of Ta4 green mold foci in commercial North American mushroom crops grown in a typical structure composed of four tiered rows made up of six stacked beds. The study revealed a non-random pattern: foci were found to occur more likely in aggregated patterns in adjacent sections along the beds, which is not suggestive of airborne contamination. In another study performed in Canada, Rinker [55] has found that only few Ta4 conidia are carried by the air. Thus in the case of the North American mushroom growing structure it is much more

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likely that inocula are spread by workers and contaminated equipment: cultural practices associated with movement along the beds (e.g. nutrient supplementation, spawning, bed tamping, surface covering) were considered as the most likely factors influencing the incidence of green mold in spawned compost [68].

5. Diagnosis of Trichoderma green mould

Because both aggressive and non-aggressive forms of Trichoderma are capable of growing in the same cultivation area [14] and remembering the difficulty to distinguish Trichoderma isolates from each other, the development of rapid, accurate, sensitive and cheap methods for identification of the aggressive Trichoderma strains was essential. This may also let the researchers know the mechanism and site of Trichoderma introduction to a farm, modes of dispersal once the infection is established, horticultural practices that allow or even promote disease, and efficacy of sanitation procedures [25]. A primer pair for the specific identification of the aggressive biotypes Ta2 and Ta4 (primers Th-F and Th-R) was developed based on a RAPD-product from Ta4 DNA in order to screen biocontrol candidates for potential pathogenicity [69]. The primer pair targets a 444 bp arbitrary sequence in the genome of Ta4 (T. aggressivum f. aggressivum), but also amplifies the same product from Ta2 (T. aggressivum f. europaeum). The specific PCR test based on these primers is useful in disease management programmes as well. Furthermore, it has been applied along with RAPD-PCR for the comparison of Trichoderma strains sampled in the United States during and prior to the outbreak of the green mould epidemic [70], which revealed no evidence for the preepidemic existence of Ta4, suggesting the recent emergence of a highly virulent genotype. The method was also applied by Hatvani et al. [33] and Szczech et al. [34] for the detection of T. aggressivum in samples from mushroom growing companies of Hungary and Poland, respectively. In the study of Hatvani et al. [33], results of the specific PCR were further confirmed by mtDNA RFLP and ITS-sequence analysis which revealed that T. aggressivum f. europaeum had geographically expanded to Central Europe. In contrast, the T. aggressivum-specific PCR gave negative results with the DNA of Trichoderma strains isolated from Hungarian oyster mushroom growing substrates affected by green mould disease [33, 71]. Sequence analysis of the ITS region, as well as fragments of the tef1 and chi18-5 (endochitinase) genes [72] supported the original findings of the T. aggressivum-specific PCR, furthermore, they revealed that similarly to the situation in South Korea, the green mould disease of oyster mushroom in Hungary was caused by T. pleurotum and T. pleuroticola [73], two species that are closely related to, but phylogenetically clearly different from T. aggressivum. Unique sequences within introns of the tef1 gene in the case of T. pleurotum and T. pleuroticola enabled the design of a multiplex PCR for the fast identification of these two Pleurotus-pathogenic Trichoderma species and their differentiation from T. aggressivum, T. harzianum and other members of the genus [64].

6. Possibilities of prevention and disease control

Green moulds develop billions of spores that are easily carried by workers, insects and contaminated equipment. Therefore, infestations are quickly spread and the control of the disease is difficult [74, 75]. Failure to treat the disease outbreaks in the early stages can be very costly as untreated areas of disease produce the spores and propagules that will spread the disease throughout the rest of the crop and the farm [76]. Poor hygiene either through ineffective disinfection of equipment or through the ingress of contaminated air into spawning halls is the likely route of the entry of the pathogen. Stringent sanitation standards coupled with the use of the proper fungicide will be required to prevent a build-up of pathogen propagules around the farm [76, 77]. It used to be a common practice for a long time to sprinkle salt over the green mould specks or to irrigate them with a benomyl containing solution. In certain cases, the whole of the infected casing material was removed and a new casing was applied [78]. In the commercial production of A. bisporus, disinfectants are frequently used as an aid to general hygiene procedure and as inhibitor of the activity of many undesirable microorganisms [79]. They are used to clean shelves, growing containers, machinery and working surface as well as floors and walls and in foot dips [80, 81]. Pasteurization of compost [82] or wood materials used in the construction of mushroom growing rooms [83] resulted in low green mould infection as well as high yield and high number of flushes. Catlin et al. [83] stated that 6 h at 60°C is adequate for the post-crop pasteurization process. However, heat treatment is not an effective preventive method because green mould pathogens have potential to survive temperatures of up to 60°C for a period of time and have been isolated from freshly pasteurized compost [84] as well. Adjustment of casing pH is another approach to Trichoderma management [85]. Attention is needed to the nature of building surface in the structures used in the mushroom industry. Greater persistence of green mould contamination was observed on the rougher surface (wood and concrete) than on the smoother glazed surface (tile) [86]. Prevention has to play a central role in green mould management, however, if the infection already occurred at a mushroom producing facility, it has to be controlled. Chemical treatments are often the most effective means of managing green mould. The minimal inhibitory concentrations of a series of fungicides towards T. aggressivum isolates were determined and compared with other Trichoderma species occurring in mushroom cultivation as well as Agaricus bisporus (Hatvani et al. unpublished), the results are presented in Table 1. Despite the expansion in the commercial

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Table 1

M

inim

al

inhib

ito

ry c

once

ntr

atio

ns

of

dif

fere

nt

fun

gic

ides

to

war

ds T. aggressivum

iso

late

s in

co

mp

aris

on w

ith o

ther

Trichoderma

sp

ecie

s o

ccurr

ing i

n m

ush

roo

m

cult

ivat

ion,

as

wel

l as

Agaricus bisporus.

Pes

tici

de

Trichoderma s

trai

ns

T. aggressivum

f. aggressivum

CB

S1

00

52

7

T. aggressivum

f. europaeum

CB

S1

00

52

5

T. aggressivum

f. europaeum

B1

T. atroviride

B1

5

T. longibrachiatum

B1

7

T. harzianum

C8

T. pleurotum

C1

5

Agaricus

bisporus

Cu

SO

4

2-1

mg

/ml

2-1

mg

/ml

1-0

.5 m

g/m

l 0

.5-0

.25

mg/m

l 0

.5-0

.25

mg/m

l 1

-0.5

mg/m

l 1

-0.5

mg/m

l 0

.25

-0.1

2 m

g/m

l

Car

ben

daz

im

1-0

.5 µ

g/m

l 1

-0.5

µg/m

l 0

.5-0

.25

µg/m

l 0

.5-0

.25

µg/m

l 0

.5-0

.25

µg/m

l >

2 µ

g/m

l >

2 µ

g/m

l >

2 µ

g/m

l

Ben

om

yl

>2

µg/m

l >

2 µ

g/m

l 0

.5-0

.25

µg/m

l 0

.5-0

.25

µg/m

l 0

.5-0

.25

µg/m

l >

2 µ

g/m

l >

2 µ

g/m

l >

2 µ

g/m

l

Thia

ben

daz

ol

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cultivation of mushrooms, only a limited number of fungicides have been recommended for application to compost, casing or spawn [87]. Initially, benzimidazole fungicides applied to the spawn gave good control of the problem but over time, mushroom pathogens like Trichoderma spp. have developed resistance to these fungicides [76, 88]. Moreover, some fungicides such as benomyl and carbendazim are known to be susceptible to microbial degradation in soils [89, 90] and this can lead to reduced control of pathogens. Several fungicides have been evaluated for controlling green mould disease and among them Environ (a commercial disinfectant) [86], Prochloraz, prochloraz + carbendazim [87] and Thiabendazol [85] were the most effective in reducing compost colonization by Trichoderma strains. Romaine et al. [91] recommended imazalil sulfate (an imidazole) against benzimidazole resistant strains. Abosriwil and Clancy [87] concluded that the placement of fungicide on the spawn gave better reduction in Trichoderma colonization than when the fungicide was dispersed throughout the mass of compost. Since the fungicides may reduce the mycelial growth of A. bisporus as well, there must be a balance between the benefit from limitation of undesirable contaminants and possible reduced vigor of the mushroom crop. Furthermore, many chemicals are no longer approved for use, and with new and evolving pathogens emerging, as well as increased demand for reduced pesticide use, growers will increasingly have to depend on disease prevention and other measures to control outbreaks rather than resorting to chemicals [76]. An alternative to chemical control of Trichoderma green mould is the application of microorganisms as biocontrol agents. Certain bacteria, including Bacillus species that naturally exist in casing are efficient antagonists of aggressive Trichoderma strains, therefore they have the potential to be used for the management of green mould disease (Fig. 2). Bhatt and Singh [92] studied the potential of antagonistic bacteria naturally occurring in casing mixtures for controlling some pathogenic fungi of A. bisporus including T. harzianum in vitro and in the mushroom beds. Among the bacterial isolates, BI III significantly controlled T. harzianum in vitro and in vivo. In addition, a higher yield was recorded in the case of T. harzianum when BI III was used against it. Győrfi and Geösel [93] studied the in vivo ability of selected bacterial antagonists (Bacillus sp.) in providing protection against T. aggressivum f. europaeum and T. aggressivum f. aggressivum infection under the growing conditions. Two strains were efficient in the control of Trichoderma species under in vivo production conditions and the bacteria enhanced both the yield and its stability.

A further option of preventing economic losses in Agaricus cultivation due to the green mould problem could be the growing of cultivars that are resistant to the aggressive Trichoderma pathogens. No defence reaction of A. bisporus cultivars to T. aggressivum attack has been observed [29, 57]. Anderson et al. [75] compared three commercial strains of A. bisporus mushrooms for resistance to green mould caused by Ta4. Hybrid white strains were extremely susceptible, hybrid off-white strains exhibited intermediate susceptibility and brown strains were highly resistant. The resistance of brown strains was also reported by Chen et al. [94]. Savoie and Mata [95] exposed A. bisporus isolates to extracellular metabolites from T. harzianum to improve their resistance to T. aggressivum (induced resistance). A. bisporus was unable to adapt to these metabolites and had a high susceptibility to T. aggressivum. Altogether, the application of appropriate and efficient prevention and control strategies against green mould is crucial in the case of Agaricus cultivation, as the infection by Trichoderma aggressivum is capable of causing serious crop losses in champignon production.

Acknowledgements The financial support of OTKA 68381, NKFP OM-00083/2004 and the János Bolyai Research Scholarship are gratefully acknowledged.

Fig. 2 In vitro inhibition of Trichoderma aggressivum f. europaeum by antagonistic Bacillus strains

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