WHAT DO WE STILL NEED TO KNOW

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Eur. J. For. Path. 19:423-434. Benjamin, D. R. 1995. Mushrooms: poisons and panaceas. New York: W. H. Freeman Co. Bertault, G., M. Raymond, A. Berthomieu ...
What Do We Still Need to Know About Commercial Production of Forest-Grown Specialty Fungi?1 Johann N. Bruhn2 ABSTRACT: Agroforestry values can be diversified and enhanced by capitalizing on natural associations of trees with specialty fungi. Fungi with potential agroforestry application include ectomycorrhizal as well as wood and litter decay species. Mushroom cultivation can provide profitable avenues for recycling low-value stemwood (either intact or in particle form) and in-place stump systems of harvested trees. The major challenges to successful mushroom cultivation lie in effectively matching specific agroforestry systems with fungi adapted to them. Areas in need of study center on fungal genetic diversity, substrate suitability and presentation, environmental constraints on vegetative growth and fruiting, integrated pest management, and development of novel uses and products. This presentation addresses the limits of our working knowledge, using as examples the fungi Ganoderma lucidum (reishi or ling chih), Grifola frondosa (maitake or henof-the-woods), Hericium species (lion’s mane and others), Lentinula edodes (shiitake), Lepista nuda (blewit), Morchella species (black and yellow morels), Pleurotus species (oyster mushrooms), Stropharia rugosoannulata (wine-cap Stropharia), and Tuber melanosporum ( Périgord black truffle).

Fungus species and even strains commonly differ in their requirements, so success in each case depends on matching compatible fungus strains with appropriate substrates under conducive environmental conditions.

Introduction Mushroom production is a natural extension of agroforestry, enhancing the overall efficiency and human interest levels of agroforestry efforts. We see these technologies as significant inducements leading to more widespread adoption of agroforestry as a land management tool. An increasingly wide variety of gourmet (Czarnecki 1995) and medicinally valued (Benjamin 1995; Hobbs 1995) mushroom species have potential for forest cultivation. The modest sample of useful fungi mentioned in this paper represents an interesting spectrum of commercial possibilities, differing in their uses and values, their ecological functions, and the relative proficiency with which we currently cultivate them. This paper considers the white-rot wood decay fungi Ganoderma lucidum (reishi or ling chih), Grifola frondosa (maitake), Hericium species (lion’s mane and others), Lentinula edodes (shiitake), and Pleurotus species (oyster mushrooms); the soil and forest floor organic matter decomposer fungi Lepista nuda (the blewit), Morchella species (the morels), and Stropharia rugosoannulata (the wine-cap Stropharia); and the ectomycorrhizal fungus Tuber melanosporum (the French or Périgord black truffle). Of these, G. lucidum can cause root disease leading to hardwood tree decline, and G. frondosa, Hericium species, and Pleurotus species are known to cause stemwood decay of living hardwood trees. The relative proficiency with which we are capable of cultivating these fungi is due in part to the limiting biological constraints unique to each species, but also to the current extent of our understanding of each mushroom species’ biology.

Outdoor mushroom production in North America is seasonal, and fruiting in-season is weather dependent except to the extent that it can be forced (e.g., certain strains of L. edodes). Most current research on mushroom cultivation focuses on indoor techniques, which permit better environmental control and more concentrated production year-round. Nevertheless, outdoor cultivation is less capital-intensive. Successful outdoor growers may expand operations to use complementary indoor practices as well, and both indoor and outdoor techniques can be fueled largely using low-value substrate materials derived from agroforestry. Though this paper focuses on outdoor production, many of the same considerations are also applicable to indoor production. Although a great deal has already been learned and written about the cultivation of specialty fungi (e.g., Singer 1961, Chang and Hayes 1978, Steineck 1981, Stamets and Chilton 1983, Przybylowicz and Donoghue 1990, Kozak and Krawczyk 1993, Stamets 1993, Elliott 1995, Oei 1996, Royse 1996b), we are still clearly on the steep portion of the learning curve. As is commonly observed in science, as we learn more we increasingly realize how little we know. This paper highlights some of our information needs in light of what we already know about the cultivation of several valuable specialty mushroom species. For this presentation, discussion has been categorized into five

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Paper presented at the North American Conference On Enterprise Development Through Agroforestry: Farming the Agroforest for Specialty Products (Minneapolis, MN, October 4-7, 1998 2 Research Associate Professor, Department of Plant Pathology, 108 Waters Hall, University of Missouri, Columbia, MO 65211, & University of Missouri Center for Agroforestry

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topics: 1) genetic diversity among species and strains; 2) substrate species form and microbiological condition; 3) environmental controls on vegetative growth and fruiting; 4) integrated pest management; and 5) novel uses and products. In summary, we can already facilitate fruiting of these mushroom species, but our progress in learning more about the biology and ecology of these and other target species will determine the extent of our future success in expanding and commercializing their cultivation.

et al. 1996). In contrast, T. melanosporum is remarkably uniform across Europe (Bertault et al. 1998), emphasizing the importance of environmental variables in helping to determine attributes such as flavor and aroma. Except for L. edodes and T. melanosporum, the mushroom species under study at the University of Missouri Center for Agroforestry are indigenous to Missouri. However, Missouri populations have not previously been studied. We are only aware of one previously studied (Pleurotus ostreatus) strain of Missouri origin (Royse and Schisler 1987), but we are currently collecting Missouri genotypes of a variety of species for comparison with commercial strains. Where vegetative strains can be obtained from local wild fruitings, they have demonstrated tolerance of the existing environment, though their other characteristics may or may not be desirable. A word of caution: the spore “rain” from a mushroom consists of genetically unique basidiospores, each the product of sexual genetic recombination. As such, they represent useful breeding material, but neither they nor the fertile strains they form upon mating possess exactly the same genetic potential as their parent vegetative isolate.

Genetic Diversity Among Species and Strains It is very useful to understand the genetic diversity among species and strains of a commercial mushroom type (e.g., the various Pleurotus species). This diversity reflects much of the genetic universe within which the tools of selection, breeding, and genetic engineering can be applied to provide strains adapted to a wider range of substrates, environments, and cultivation methods (Kapoor et al. 1996, Ikegaya 1997). Greater genetic diversity also suggests greater available variance in mushroom yield characteristics such as mushroom size, color, shape, flavor, and texture, as well as season of fruiting and biological efficiency (BE, a measure of the efficiency with which a strain converts substrate mass into mushroom mass). The potential for breeding or engineering “sporeless” mushroom strains (e.g., of P. ostreatus: Leal-Lara 1978, Imbernon and Laberere 1989) which circumvent concerns over respiratory allergic reactions, especially among mushroom industry workers, is another significant potential use of genetic diversity.

Relationships are being developed which link key metabolic characteristics of mushroom strains to their relative growth rates, mushroom development, and perhaps ultimately their BE. For example, one study has correlated Pleurotus strain productivity with laboratory growth rate in the presence of the secondary metabolite 2-deoxy-D-glucose (Sanchez et al. 1996). Other studies have drawn attention to possible links between production of lignin-degrading enzymes and vegetative growth and fruiting by Pleurotus species (Das et al. 1997, Eichlerová-Voláková and Homolka 1997, Homolka et al. 1997) and L. edodes (Leatham and Stahmann 1981). Techniques developed in these types of investigation could eventually prove useful for breeding and/or screening candidate strains (e.g., Mata et al. 1998).

A variety of strains of G. lucidum, Hericium erinaceus (lion’s mane), L. edodes and Pleurotus species are commercially available which yield high value products for which markets already exist (Stamets and Chilton 1983, Leonard and Volk 1992, Kozak and Krawczyk 1993, Stamets 1993). Availability of isolates and cultivation techniques for G. frondosa, other Hericium species, L. nuda, Morchella species, and S. rugosoannulata are still arguably in a more primitive state (Garbaye et al. 1978, Stamets and Chilton 1983, Stamets 1993, Stott et al. 1996), helping to explain why their markets are under-developed even though their qualities are widely recognized. Nevertheless, cultivation efforts with all the above species would benefit from further exploration of their genetic diversity for strain selection and breeding (e.g., Fritsche and van Loon 1989, Guinberteau and Olivier 1991). For example, the magnitude of genetic

Substrate Species, Form, and Microbiological Condition Though all the mushroom-producing species considered here originated in the natural forest setting, their relative adaptability to various substrate species and forms (e.g., stumps, logs, wood particles, leaf litter, etc.), and their preferences with respect to the microbiological condition of their substrates, are not completely clear.

variation within Morchella species rivals that between species (Bunyard et al. 1994). Also, intensive study has led to the recent distinction of a number of new Pleurotus species native to North America (Vilgalys 151

Substrate Species

damaged by the eastern filbert blight fungus (Anisogramma anomala) and powdery mildews, respectively, than are native Corylus and Quercus species. For this reason, it will be important to test hybrid genotypes of both Corylus and Quercus as hosts for use in North American T. melanosporum truffières.

The North American host tree species preferences of many of these fungi are not clearly understood from the perspectives of vegetative growth rate and fruiting in mushroom cultivation systems. This should not be surprising for such fungi as certain G. lucidum strains (Okamoto and Mizuno 1997), L. edodes (Przybylowicz and Donoghue 1990), some Pleurotus species (Date 1997), and T. melanosporum (Bertault et al. 1998), which are not native to North America. While the relative productivity of L. edodes on different log species has been fairly well established, little is known about L. edodes production possibilities on outdoor beds of different species of wood chips, an intriguing method practiced in the Peoples Republic of China (Krawczyk, personal communication). Ganoderma lucidum, G. frondosa, L. nuda, Pleurotus species, and S. rugosoannulata are also capable of utilizing a variety of hardwood species as substrate (Steineck 1981, Stamets and Chilton 1983, Przybylowicz and Donoghue 1990, Kozak and Krawczyk 1993, Stamets 1993, Date 1997), and even T. melanosporum is capable of using a variety of tree species as ectomycorrhizal hosts (Hall et al. 1994). Different species and strains of these fungi certainly must grow and/or fruit differently in association with different substrate/host species, and knowledge of these relationships could guide agroforesters in species and strain selection. For just one example, productivity of L. nuda and S. rugosoannulata on Quercus vs. Acer mulches and cereal straw should be compared. Field experiments can be laid out as Latin squares in complete blocks where possible (Sokal and Rohlf 1995). Fruit body production by candidate strains of each fungus growing on different substrate species can be compared by Analysis of Variance (or Multiple Analysis of Variance), using BE with or without other measures of mushroom size or quality as dependent variables.

Substrate Form The suite of fungi cultivated in each agroforestry situation should be based on knowledge of strain productivity on the available substrate form(s) as well as species. Fungus strains (e.g., strains of G. lucidum, G. frondosa, L. edodes, and Pleurotus species) may rank differently in productivity on different substrate forms (e.g., stumps, logs, wood chips). Different substrate forms present fungi with different moisture and aeration levels, as well as offering the opportunity to add nutrient supplements (Royse and Schisler 1987, Royse 1996a). While bagged wood chip or sawdust substrates are usually used for indoor culture, some fungi also fruit easily outdoors from bagged substrate (Cha and Yoo 1997, Mayuzumi and Mizuno 1997). It has been suggested that logs cut from living trees for G. lucidum (Mayuzumi et al. 1997) and L. edodes cultivation (Ikegaya 1997) should be aged for at least a month after tree felling before inoculation. The rationale for this method is that spawn run should be faster in aged logs because both fungi should grow faster through substrate logs which have been allowed time to die completely. However, as logs age and wood cells die, they become accessible to antagonistic fungi, especially if they are in soil contact and/or are allowed to dry out (Przybylowicz and Donoghue 1990, Kozak and Krawczyk 1993). Pre-colonization of substrate logs by antagonistic fungi naturally reduces access to the logs by L. edodes and shortens the logs’ useful life. Compared with L. edodes, Pleurotus species, and S. rugosoannulata, Gramss (1978) found that G. frondosa was less capable of colonizing fresh-cut logs unless they were sterilized, raising questions about the effectiveness of cultivating this fungus on fresh hardwood stumps. Outdoor cultivation of G. frondosa in Japan commonly employs sterilized blocks of sawdust amended with corn and wheat brans (Mayuzumi and Mizuno 1997). Even Pleurotus ostreatus can require three growing seasons to fruit following stump inoculation (Krawczyk and Kozak, personal communication). The difficulty with which non-parasitic decay fungi colonize fresh stumps may depend on the extent to which they are root grafted to surrounding live trees; stump root systems kept alive by root grafts can be expected to maintain some

Ectomycorrhizal fungi such as T. melanosporum depend principally on living host trees for carbohydrate, in return for which the fungi provide their host tree’s root system with scarce soil minerals and water. Productive T. melanosporum truffle orchards (truffières) have now been established outside the fungus’ native range, in New Zealand (Hall et al. 1994) using Corylus avellana (European filbert) and Quercus robur (English or truffle oak) and in North Carolina (Garland 1996) using C. avellana. Although the fungus is capable of utilizing a wide variety of tree species as hosts (Hall et al. 1994), commercial ventures have favored C. avellana for its shallow rooting habit and rapid growth, and Q. robur for its longevity. In North America, however, C. avellana and Q. robur are much more severely 152

resistance to spawn run in the inoculated stump. Stump inoculation techniques may be more effective in stands of mixed tree species composition, where root grafting would be less likely between neighboring trees.

availability of decaying organic matter (e.g., dead root material) can be inferred from the association of fruiting with abandoned fruit orchards, dead elm trees, fires, etc. (e.g., Volk 1991, Leonard and Volk 1992, Stamets 1993).

Microbiological Condition

Cultivation of T. melanosporum is also a challenging prospect. As an ectomycorrhizal fungus, T. melanosporum is probably the most fastidious species mentioned here. The black truffle has not been grown in Missouri, but neither has its cultivation been attempted anywhere in the Midwest. Conducive soil and climate conditions appear to occur in central Missouri, and truffle cultivation would be of tremendous commercial value. The challenges associated with growing T. melanosporum are perhaps less due to lack of biological understanding, and more associated with attempting to provide host and soil conditions appropriate for development and fruiting (Singer 1961, Delmas 1978, Hall et al. 1994, Callot and Jaillard 1996, Garland 1996, Chevalier and Frochot 1997). This fungus prefers well-drained alkaline soils with balanced texture developed in contact with calcareous bedrock. Because T. melanosporum competes poorly with many indigenous mycorrhizal fungi (e.g., Scleroderma species), truffières should be established on sites which have not been forested for some time. The postulated protective effect of soil pseudomonad bacteria favoring T. melanosporum colonization of hazel roots over competitor fungi (Mamoun and Olivier 1992) and the observed association of larger truffles in the proximity of Festuca ovina within the brûlé (Mamoun and Olivier 1997) need to be studied.

It has long been known (e.g., Mayzumi et al. 1997) that fungi range from relatively intolerant of competition from other microbial colonists (e.g., G. lucidum, G. frondosa) to more or less dependent on microbial pre-colonization as a source of nutritional factors or to produce a conducive environment for fruiting . Depending on the species, fruiting (e.g., G. frondosa, L. nuda, Pleurotus species, and S. rugosoannulata) may be enhanced by substrate composting or sterilization, with or without organic amendments (Stamets and Chilton 1983, Gramss 1989, Stamets 1993, Oei 1996). Technique of substrate incubation in the field (e.g., partially buried vs. surface incubation of logs or wood chips) affects productivity of G. lucidum, G. frondosa, and Pleurotus species through effects on substrate moisture content as well as stimulatory microbial colonization (Omori 1974, Stamets 1993). Environmental Controls on Mycelial Growth and Fruiting Soil Factors Cultivation of Morchella species is one of our most challenging research prospects, even though morels are common in Missouri. From a cultivation standpoint, we probably know less about the critical field biology of Morchella species than any of the other species mentioned here. We are not aware of any reproducible system for outdoor commercial culture of morels, and patented techniques for indoor cultivation of morels have fallen short of expectations. Factors favoring efforts to cultivate Morchella species outdoors include abundant local genotypes from which to select effective strains, the ability to produce pseudosclerotia in culture (Volk and Leonard 1990, Volk 1991, Leonard and Volk 1992, Stamets 1993, Philippoussis and Balis 1995), and the ability to sample pseudosclerotia in the field (Miller et al. 1994). Morchella pseudosclerotia can be used to establish statistically rigorous field studies. Challenges include: development of appropriate field inoculation techniques; identification of appropriate soil conditions and substrata for mycelial growth, pseudosclerotium development, and fruiting; and development of techniques for providing these appropriate conditions perennially (Stamets 1993, Faris et al. 1996). Dependence of morel fruiting on

It has been reported (Garbaye et al. 1978) that forest fertilization with NPKCa mineral fertilizer induced abundant fruiting of valuable mushroom species which normally would not be found on the sites involved (including L. nuda and the ectomycorrhizal species Boletus edulis). It is intriguing to speculate whether fertilization provided conditions for these species to fruit on a site they already occupied, or provided conditions which permitted these fungi to invade and flourish vegetatively at the involved sites as well. Casing Requirements Some fungi fruit more abundantly when the colonized substrate is covered (“cased”) with a water-retentive soil-like material. For some of these fungi, effective casing materials contain stimulatory bacterial microflora (Singer 1961, Stamets and Chilton 1983, Oei 1996). Fungi which benefit from microbial substrate conditioning (e.g., G. frondosa, G. lucidum, L. nuda, and S. rugosoannulata) respond differentially to various casing materials (Szudyga 1978, Stamets 153

and Chilton 1983, Upadhyay and Sohi 1989, Stamets 1993, Sharma et al. 1996). For example, BE values achieved by fungus strains (even strains of the same species) vary with and without pH-adjusted peat-based or soil-based casing. The responses of these fungi to casing techniques needs to be clarified.

capable of killing hardwood trees of a wide range of species (Sinclair et al. 1987). Although the distribution patterns of these fungi in forests do not suggest tree-to-tree spread across root grafts or contacts, spread may be accomplished through colonization of basal or root wounds by airborne spores produced by the mushrooms. The prospects, in general, for root disease leading to tree decline associated with tree root damage caused by farming equipment in alley cropping systems and grazing animals in silvopastoral systems merit study. Hericium species, L. edodes, and Pleurotus species are known to cause decay of dead wood in stems and branches of living trees. Silvicultural care to prevent stem and root wounding of residual trees coupled with techniques for efficient production of these fungi on stumps, logs and wood chip mulch, would greatly increase production while reducing concerns over spread of root disease or heart rot.

Determinants of Fruiting Harvesting is most efficient when flushes of mushrooms can be planned and induced. BE may be improved if flushes can be induced early, thus utilizing the substrate while it is in best condition and reducing substrate loss to competing microbes. Water management techniques are useful in controlling flushes of some L. edodes strains (Przybylowicz and Donoghue 1990, Kozak and Krawczyk 1993, Tokimoto et al. 1998). It may also be possible to identify differences among strains of fungi in season of fruiting (e.g., Pleurotus species) or amenability to controlled flushing (e.g., S. rugosoannulata) which can be used to level out mushroom production over time.

Mushroom Diseases, Insect and Animal Pests, and Weed Fungi Prominent disease and/or competitor fungi in log culture of mushrooms include Trichoderma, Diatrype and Hypoxylon species, and Armillaria, Lenzites, Stereum and Trametes species, respectively, among others. Both incubation of substrate in soil contact and allowing substrate to dry out raise the probability of losses to these fungi (Przybylowicz and Donoghue 1990, Abe 1989, Kozak and Krawczyk 1993). If “aging” of substrate logs prior to inoculation results in faster spawn run, then logs must also become increasingly vulnerable to colonization by antagonistic fungi during the aging process. Research should address the need to produce conditioned substrate logs for fastest possible spawn run while minimizing antagonistic microbial contamination.

Mushroom form can be at least as important as BE, especially when mushrooms are grown for their artistic value. Levels of moisture, light and carbon dioxide around developing crops of mushrooms exercise substantial influence over mushroom form for G. lucidum (Stamets 1993, Chen and Miles 1996). Gas exchange during incubation as determined by culture bag filter patch size affected total L. edodes mushroom yield, mushroom size and number, as well as degree of contamination, and the optimum level of gas exchange differed among the strains tested (Donoghue and Denison 1995). Tokimoto et al. (1998) have demonstrated that the extent of fruiting by L. edodes from bedlogs of Quercus serrata is determined by the balance achieved between free water content and air volume during soaking, and that the desirable free water content increases greatly as bedlog decay progresses. This relationship needs to be further characterized for various bedlog species and L. edodes strains.

Amendments which may enhance yields can also influence microbial populations, leading to contamination problems (Houdeau et al. 1991). While both formaldehyde treatment of nutritional supplements (Houdeau et al. 1991) and fungicide treatment of substrates (Lelley and Niehrenheim 1991) have been used to prevent substrate contamination, clean technique and providing a physical environment which favors the cultivated strain are more desirable cultural means of controlling contamination. Use of pesticides should be avoided, with emphasis placed on cultural means of control. Helpful techniques include keeping substrate units out of soil contact when appropriate, creating a gravel layer under substrate units, promptly harvesting crops, and avoiding the temptation to force harvests during cool weather (Przybylowicz and Donoghue 1990).

Integrated Pest Management Tree Disease Of the fungi we currently propose to cultivate in the UM Agroforestry Center, only G. lucidum merits attention as a possible agent of tree root disease and decline. Both G. lucidum and G. frondosa are known to cause white heart rot of the butt and root portions of living and dead hardwoods of various species (Farr et al. 1989). Grifola frondosa is not reported to cause tree decline or mortality, but Ganoderma lucidum is 154

Novel Uses and Products

Literature Cited

Mushroom production offers a unique opportunity to bio-convert agricultural and forestry waste materials into valuable foods and medicines. Although the lignocellulosic components (cellulose, hemicellulose, and lignin) of plant structure are the most abundant organic materials in the biosphere, their complexity and arrangement in plant cells (especially lignin) makes it extremely difficult for animals to digest them (Ohga and Kitamoto 1997). Although the products of interest for human consumption are the mushrooms, the mycelia which give rise to mushrooms release enzymes into their substrate which decompose lignocellulose components to simpler substances. In doing so, fungi including some Pleurotus species and S. rugosoannulata can raise the overall digestibility of wheat straw substrate, suggesting that methods may eventually be developed for producing useful animal feed from spent mushroom-growing substrate (Zadrazil 1978, Kapoor et al. 1996). Thus, improvements in the enzymatic efficiency of mushroom-producing fungi through breeding or genetic manipulation may simultaneously improve mushroom yields while generating useful animal feed products.

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