Introduction: Globalization of the Mushroom Market

Many wild and cultivated mushrooms have longbeen consumed for their high protein content (crudeprotein represents roughly 20–30% of the dry matter)and rich B vitamins, but they also have low fat and arevirtually free of cholesterol. Fungal biomass is digestedwith their chitinous wall left as dietary fiber. Mush-rooms are labeled as natural and healthy food prod-ucts originating from an environmentally friendly or-

ganic farming system (Moore and Chiu, 2001). Fur-thermore, there are the wild collected mushrooms, in-cluding truffles, whose unit price is even higher thangold; the most highly prized in French cuisine is theblack truffle of the Périgord, Tuber melanosporum(Molina et al., 1993). And the world market forchanterelles was estimated recently at more than US$1.5�109 for 150,000–200,000 Mt (Watling, 1997). Thesecrops from nature are usually treasured for their rarityor unique appeal such as flavor or taste, or both (Jongand Birmingham, 1993; Pilz and Molina, 1996).

Agaricus bisporus (the common cultivated mush-room, also called the button mushroom) and its relatedspecies are still the most commonly cultivated mush-rooms throughout the world. Global Agaricus mush-

Themes for mushroom exploitation in the 21st century: Sustainability,waste management, and conservation

Siu-Wai Chiu,* Shui-Chee Law, Mei-Lun Ching, Ka-Wan Cheung, and Ming-Jie Chen

Department of Biology and Environmental Science Programme, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China

(Received November 2, 2000; Accepted December 9, 2000)

Because many natural resources are limited, sustainability becomes an important concept inmaintaining the human population, health, and environment. Mushrooms are a group of sapro-trophic fungi. Mushroom cultivation is a direct utilization of their ecological role in the biocon-version of solid wastes generated from industry and agriculture into edible biomass, whichcould also be regarded as a functional food or as a source of drugs and pharmaceuticals. Tomake the mushroom cultivation an environmentally friendly industry, the basic biology of mush-rooms and the cultivation technology must be researched and developed. This is very true forLentinula edodes, Volvariella volvacea, and Ganoderma lucidum, which are commonly con-sumed in Asian communities but are now gaining popularity worldwide. Besides the conven-tional method, strain improvement can also be exploited by protoplast fusion and transforma-tion. Biodiversity is the key contribution to the genetic resource for breeding programs to fulfilldifferent consumer demands. The conservation of these mushrooms becomes essential and isin immediate need not only because of the massive habitat loss as a result of human inhabita-tion and deforestation, but also because of the introduced competition by a cultivar with the wildgerm plasm. Spent mushroom compost, a bulky solid waste generated from the mushroom in-dustry, however, can be exploited as a soil fertilizer and as a prospective bioremediating agent.

Key Words——bioconversion; conservation; exploitation; mushroom; spent compost; sustainability

J. Gen. Appl. Microbiol., 46, 269–282 (2000)

* Address reprint requests to: Prof. Siu-Wai Chiu, Departmentof Biology, The Chinese University of Hong Kong, Shatin, N. T.,Hong Kong, China.

E-mail: siuwaichiu@cuhk.edu.hk

Invited Review

room production shows an increasing trend reaching2,383,710 Mt in 2000 (National Agricultural StatisticsServices, U. S. Department of Agriculture [NASS,USDA], http://usda.gov.nass; Statistical Data, Foodand Agricultural Organization of the United Nations[FAO], http://apps.fao.org) (Fig. 1). This accounts for30% of the world mushroom production. Globalizationand efficient transportation enhance the importing ofproducts from other countries; thus the price per unitweight has dropped significantly over the years, butthe demand for good-quality products has been in-creased. For similar reasons and as a result of masshuman migration and cultural exchange, even the so-called exotic/specialty mushrooms such as the Lentin-ula edodes and Pleurotus species have penetratedmarkets worldwide. The market for the mushroombusiness has been the very source of finance to sup-port basic research on mushrooms, especially in re-gard to cultivation, fruiting, and genetic breeding sys-tems. In the 21st century, however, it is predicted thatmushroom science will broaden its focus. The exploita-tion of mushrooms and their products for other uses,especially environmental protection, should be empha-sized on one hand. But on the other, conservation ofthe biodiversity of mushrooms and a basic understand-ing of mushroom biology should be researched, devel-oped, and emphasized for the sustainable uses ofmushrooms and their products.

1. Mushroom Cultivation Can Be a Profitable, Sus-tainable, and Environmentally Friendly Solid-stateFermentation

China is the champion mushroom producer and ex-porter for many mushroom species, including A. bis-porus, L. edodes, and Ganoderma lucidum (Mooreand Chiu, 2001; http://www.americanmushroomist.org;http://apps.fao.org). For the export of Agaricus spp.,however, other countries—including Mexico, theNetherlands, and India—are catching up (Anonymous,1999; Tschierpe, 1996). The commonly called Chinesemushroom refers to Volvariella volvacea, the paddystraw mushroom, which has the longest cultivation tra-dition, or L. edodes (common names: black oak mush-room, shiangxu, and shiitake), which is found in Asia-Australasia (Hibbett et al., 1998; Shimomura et al.,1992; Tokimoto et al., 1973). The latter was first culti-vated in China (Chang and Lai, 1993), but it has beenconsumed and examined as a favorite by the Japa-

nese (Ikegaya and Goto, 1988; Ishizaki et al., 1999;Katayose et al., 1992; Miyazaki et al., 1997; Nakai,1986; Shimomura et al., 1992).

The Western world has focused on Agaricus for con-sumption. This focus therefore leads to advancementin cultivation technology from farm design, quality con-trol in compost production, microprocessor control andrecords for growing and mechanical harvesting, andenvironmental guidelines in mushroom production(http://www.agro.nl/pc/Pceng.htm; The mushroommanual, http://www.dep.state.pa.us). The Netherlands,practising a high-technology cultivation system, thushas the highest mushroom yield per unit area(http://apps.fao.org) (Fig. 2). This machine-based culti-vation also reflects the high cost in manpower in theseWestern countries. In the Far East, however, mush-room cultivation is usually rural in practice and takesadvantage of cheap labor instead of making the greatinvestment required by expensive instruments and asophisticated cultivation schedule (Moore and Chiu,2001). In these countries, mushroom cultivation is stillregarded as an art more than it is a science becauseof the flexibility in human adjustment and very exten-sive experience.

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Fig. 1. The production trend of the edible button mushroomAgaricus by the top 10 countries from 1995 to 2000 (Datasource: Food and Agricultural Organization of the United Na-tions, http://apps.fao.org).

Some countries maintain their production tonnages, butChina shows the greatest trend of increase. The global mush-room market is expanding.

All in all, commercial mushroom production is asolid-state-fermentation technology. All agriculturalproduction for plant crops generates enormous wastebecause so little of each crop is actually used; typically80–90% of the total biomass of agricultural productionis discarded as waste (Chang, 1999). Landfilling withthis abundant waste is costly and ineffective in termsof energy flow, and combustion is indeed a waste ofresource, though some nutrients in the straw can bereturned to the agricultural land if the straw is burnedin situ. The biological efficiency of a cultivated mush-room strain (in terms of fresh mushroom yield over thedry weight of the compost used) ranges from 17 to250% (Chang, 1993; Chang and Chiu, 1992; Pani etal., 1998). Thus mushroom cultivation can usually beviewed as an effective means to extract resources leftbehind in agricultural solid wastes. This waste biocon-version process is simultaneously also a sound envi-ronmental protection strategy too.

The fast-growing oyster mushrooms of genus Pleu-rotus, having a complete lignocellulolytic enzyme sys-tem, unlike the nonlignolytic V. volvacea, can use awide spectrum of agricultural and industrial wastes forgrowth and fruiting (Chiu et al., 1998a; Hadar et al.,

1993; Ortega et al., 1992). Moreover, in the genusPleurotus different species are suitable for cultivationfrom low to high temperatures, respectively (http://www.americanmushroomist). Therefore besides itshigh biological efficiency and ease in cultivation, oystermushroom production experienced an increase ofmore than 200% from 1985 to 1991 (Chang, 1993). V.volvacea is traditionally grown on rice straw and nowcommonly is grown on cotton waste. Mushroom culti-vation has thus been exploited to treat industrialwastes, including sawdust and wastepaper (Changand Chiu, 1992; Chiu et al., 1998a).

The Agaricus cultivation bioconverts animal wastes,including chicken manure and horse manure. Thusammonia and sulfur-rich compounds are nuisance airpollutants generated from composting. The humannose can sense 10 ppm ammonia, but the ammoniaemitted from fermenting compost can reach 600 to1,000 ppm. This pushes toward the development of anindoor composting system in which generated air pol-lutants are collected and converted to harmless am-monium sulfate, which can later be sold (http://www.cnc.nl/nutshell.html). Alternatively, a composting de-sign with a pipe of multiple outlets for fresh air to flowin and out of a pile of mushroom substrate has recently been developed to prevent the accumulationof odorous airborne pollutants generated from anaer-obic decomposition (http://aginfo.psu.edu/PSA/s99/mushroom6.html). The cultivation of other mushrooms,however, depends on composted plant litter or rawmaterials without composting; thus the release of odor-ous air pollutants will not be as great.

The harvest of dried L. edodes in 1997 wasrecorded as 91,500 Mt (100,833 tons) in China and accounted for about 70% of world production (vanNieuwenhuijzen, 1998; Wang, 1998). The traditionallog-pile cultivation method, however, leads to a severethreat to natural forests; up to 100,000 trees must befelled every year just to maintain current productionlevels. When mature trees decline in amount, irrespon-sible farmers turn their attention to young trees. Thisoutdoor wood-log cultivation system threatens China’sforests. A sustainable mushroom industry requiresmanaging tree plantations for the timber industry, pro-tecting forests, and recycling the timber wastes formushroom cultivation. It requires involvement of thepeople for protection of the land and for proper use ofresources for living to make mushroom cultivation en-vironmentally friendly (Chiu and Moore, 2001).

2000 Mushroom exploitation in the 21st century 271

Fig. 2. The trend of Agaricus production effectiveness bythe top six countries in comparison with the world average forthe years 1995 to 2000 (Data source: Food and Agricultural Or-ganization of the United Nations, http://apps.fao.org).

Production effectiveness measures the mushroom yield perunit area. Although China has the highest mushroom productiontonnage, its production effectiveness is below the world aver-age. The Netherlands practicing a high-technology mushroomfarming system has the highest production effectiveness.

Over the past several years, we have investigatedthe population biology of L. edodes in the wild and incultivation (Chiu et al., 1999, unpublished results) (Fig.3A). The experimental evidence suggests that differentisolates exist in a wood log in the wild and also inmushroom farms whose owners only artificially inocu-late a single cultivar to the wood logs. L. edodes uti-lizes basidiospore dispersal for species propagation.Random mating events among the haploid ba-sidiospore germlings in a wood log take place in thewild, and this leads to several individuals in a log (Chiuet al., 1999) (Fig. 3A). In a mushroom farm that prac-

tises the outdoor wood-log system, L. edodes mush-rooms are harvested after the partial veil is open, ex-posing the maturing gills. Basidiospores then becomea contaminating source and lead to an unpredictableyield in later flushes as a result of genetic heterogene-ity instead of strain degeneration (Chiu et al., 1999).Furthermore, an outdoor mushroom farm system alsocreates a great source for spreading the genome ofthe cultivar, which thus dilutes and competes with thewild germ plasm (Hibbett and Donoghue, 1996). Boththe wild isolates from southeast China and the Japa-nese cultivars have the rDNA sequence of lineagegroup 1 (Chiu et al., 2000b; Hibbett et al., 1998), andthe Japanese cultivars were imported to various coun-tries for mushroom production and used as parent forstrain improvement programs (Chiu et al., 2000b; Hib-bett et al., 1998; Chang and Lai, 1993). It might there-fore not be surprising that the rDNA lineage group 1has the greatest distribution, covering Japan, Korea,China, and Thailand (Chiu et al., 2000b). To reducethe threat to wild germ plasm, breeding work might target for cultivars, which are spore-deficient or spore-less or are domesticated native wild isolates. Or amarketing strategy might need to convince consumersto accept younger fruiting bodies and push the industryto harvest immature mushrooms. Nowadays the high-price market for A. bisporus is for the fresh small but-ton mushrooms (miniature fruit bodies still at an imma-ture stage) and not the large ones (Moore and Chiu,2001).

Care must be exercised, however, because manymushrooms accumulate metal ions and radioisotopesin the fruit bodies. Wastes gathered from industrial orpolluted agricultural sources for use in mushroomcompost may be contaminated by heavy metals suchas Hg, Cd, and Pb to an extent sufficient to render thecrop unsuitable for consumption (Brunnert andZadrazil, 1983; Fischer et al., 1995). For instance,Chiu et al. (1998a) showed that cadmium could be ac-cumulated in Pleurotus fruit bodies to such high levelsthat a single modest serving of mushrooms couldcause the consumer to exceed the tolerable food limitrecommended for a full week of intake of this metal.On the other hand, utilizing the mushroom bioaccumu-lation capabilities, certain beneficial minerals, e.g., cal-cium and selenium, are added to the compost to en-hance the nutritive value of the resultant mushroomcrops (Chiu et al., 1998a; Hartman et al., 2000).

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Fig. 3. Population structures of (A) Lentinula edodes and(B) Ganoderma lucidum observed in the field.

(A) A fruiting body represents an individual and is repre-sented as a strain number. (B) All fruiting bodies (representedfrom a to k) on and around a tree belong to the same individual.The identity of an individual is based on a somatic incompatibil-ity test, DNA fingerprints, and colony morphology.

2. Diversification of Mushroom Products

Langley (1997) reported the 20 top-selling prescrip-tion medicines in 1996, and six compounds were offungal origin. Three closely related compounds,namely, pravastatin, simvastatin, and lovastatin, all de-rived from mevinolin showing hypolipidemic activity,had a sale of US$4.94 billion in 1995. Mushrooms area rich source of these bioactive compounds (Sugiyamaet al., 1993; Wasser and Weis, 1999). The Chineseclassic medical book “Shen Nong Ben Cao Jing,” writ-ten at AD 25–200, has described 18 mushroomspecies as herbal medicines. Since then the numberhas increased to more than 50, and these are con-sumed as traditional Chinese medicines (Anonymous,1998). With the spread of Asian cultures worldwide,food is consumed not only for its nutrition, but also forother beneficial effects, especially the medicinal prop-erties as introduced by the concept of health food,functional food and nutraceuticals (nutrients that havesome additional medical value) (Anonymous, 1998;Mizuno et al., 1995). For instance, many research pro-jects show that mushroom polysaccharides have vari-ous medicinal properties, including antiaging, antioxi-dant, antiviral, anticancer, hepatoprotective, and neu-roactive properties (Anonymous, 1998; Cheung et al.,2000; Wasser and Weis, 1999). But their action mech-anisms must be elucidated. Proper scientific researchoften may not be associated with the beneficial claimby some commercial mushroom products (Chiu et al.,2000c). Furthermore, with the inherent complexity andpotential interaction among various compounds in aherbal medicine such as mushroom extracts, and theextent of bioavailability of the effective agents after di-gestion, it is difficult and may take more than 10 yearsto develop a mushroom pharmaceutical. Thus the mar-ket goes to functional foods or nutraceuticals. Dietarysupplement sales in U.S.A. were more than US$6.5billion in 1996 (http://vm-cfsan.fda.gov). In 1994,US$3.6 billion was cited as representing the amountgenerated from sales of mushroom products, includingtonics and medicinal (Chang, 1999). Under the nameslingzhi or reishi, several Ganoderma species providevarious commercial brands of nutraceuticals. Thus theglobal production of Ganoderma in 1997 reached3,902 Mt (4,300 tons) (http://www.kyotan.com).

In a harvested mushroom crop, not all parts areused for consumption. For instance, cleaning andchopping of the basal stem part of an Agaricus mush-

room (to remove the compost trace) are carried out. Ifmechanical harvesting is practised, the wasted mush-room parts could exceed 50%, but these parts can beturned into profit. Chitin, a fungal cell wall component,has various applications, including the sorption ofheavy metals and cosmetics (Chung et al., 1998; Joenet al., 2000; Kapoor and Virarahavan, 1995; Kratochviland Volesky, 1998; Volesky, 1990). The waste mush-rooms can be a readily available source in steady sup-ply for chitin extraction. Our unpublished study indi-cates that mushroom-extracted chitin and commercialcrustacean chitin actually have a similar performancein metal adsorption, and the isolated mushroom wallalso performs better than the fungal biomass (Fig. 4).Besides, mushroom colonized substrates have beenused as animal feed and agents for biodegradingorganopollutants (Wolter et al., 2000; Yateem et al.,1998).

3. Spent Mushroom Substrate: A Potential Bioreme-diation Agent

For each metric tonne of mushrooms produced, atleast an equivalent amount of spent mushroom sub-

2000 Mushroom exploitation in the 21st century 273

Fig. 4. A comparison of the removal efficiencies of ex-tracted mushroom chitin (�), extracted mushroom wall (�), andmushroom (�) of Pleurotus pulmonarius toward cadmium.

Experimental conditions: 20 mg of freeze-dried and powderedmaterials were incubated in 1.2 ml of cadmium solution in anacid-treated Eppendorf tube at 150 rpm by using a roller drumat ambient temperature for 2 h. The residual and initial cadmiumconcentrations were measured by using atomic absorptionspectrometry. Removal efficiency means the percentage ofmetal removed over the initial amount of metal used. The dataare represented in a mean and standard deviation of 5 repli-cates.

strate (also called spent mushroom compost) is gener-ated, and this needs to be disposed of! This disposalcan be costly because it is bulky, and in developedcountries such as the U.K., disposal charges andtaxes on landfill are now implemented (Fermor et al.,2000). Attempts have been made to recycle or reusethis waste (Sharma et al., 1999). Spent mushroomcompost, because of its high mineral contents, phos-phate content, and high porosity, is a good soil condi-tioner and soil fertilizer for stimulating seed germina-tion (Ching, 1997; Szmidt, 1994). On the other hand,the mushroom crops release abundant extracellularenzymes during growth and fruiting to digest the sub-strate (Hammond, 1981; Turner et al., 1975). Themushroom species does affect the quality and quantityof enzymes immobilized in the spent compost. Thisspent substrate can be a good source of lignocellu-lolytic enzymes for biodegrading various xenobioticsand persistent pollutants, including pentachlorophenol,polyaromatic hydrocarbons, and azo dyes (Chiu et al.,1998b, unpublished result; Fermor et al., 2000; Harm-sen et al., 1999) (Fig. 5). This waste substrate alsohas a good pH buffering capacity, since lime is addedduring preparation of the mushroom compost; thus thespent compost can be used to remedy acidic minewastewater (Wieder, 1993). Furthermore, spent mush-room compost has many different functional groupsfrom mushroom mycelia and substrate lignocelluloseto act as sorbent sites for various metallic and organo-pollutants (Ching, 1997; Chiu et al., unpublished re-sult) (Figs. 5 and 6). Moreover, the spent mushroomsubstrates also harbor a consortium of diverse bacte-ria and fungi, which besides the immobilized enzymeshelp the biodegradation of organic pollutants (Ching,1997; Chiu et al., unpublished result; Wiesche et al.,1996) (Fig. 6). Also the composting of solid waste andrefuge materials is a common way to generate energyin rural areas. Spent mushroom compost actuallyspeeds up emission of methane biogas (Ching, 1997;Sharma et al., 1989).

4. Fruiting Morphogenesis, Phenotypic Plasticity,and Biodiversity

Traditional taxonomy emphasizes the morphologicalfeatures to characterize a taxon. However, develop-mental plasticity is actually an adaptive feature to thefluctuating and unpredictable environment (Chiu andMoore, 1996, 1999a; Chiu et al., 1989; Moore, 1998).

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Fig. 5. The removal efficiencies of polyaromatic com-pounds (PAHs) singly (A) and in combination (B) by the spentmushroom compost of Pleurotus pulmonarius.

Symbols: �, biodegradation; �, biosorption. Experimentalconditions: 100 mg of freeze-dried and powdered spent mush-room compost were incubated in 10 ml of single or a mixture ofPAH(s) solution in a 50 ml Falcon tube at 150 rpm by using aroller drum at ambient temperature overnight. The spent mush-room compost was harvested by centrifugation at 13,000 rpmfor 20 min. The PAH contents in the supernatant, the pellets ofthe spent mushroom compost, and the initial mixture were de-termined after extraction with dichloromethane (HPLC grade)and quantified by using the authentic standard(s) by gas chro-matography-mass spectrometry. Biosorption refers to the de-tected PAH amount sorbed by the spent mushroom compost.Biodegradation means the difference between the initial PAHamount used and the amounts of PAH found in the spent mush-room compost and in the supernatant. Removal efficiency is ex-pressed in percentage of the removal by a particular mecha-nism over the initial PAH amount used. The data are repre-sented in mean and standard deviation of 5 replicates.

Like Volvariella bombycina (Chiu and Moore, 1993;Chiu et al., 1989), Ganoderma also expresses muchdevelopmental and morphological diversity (Chiu et al.,2000a). When the habitat is exposed to direct sunlight,the mushroom has a dark red shiny surface; when themicrohabitat is sheltered or the nutrition is poor, thecolor can remain as pale yellow; in the field, an individ-ual produces different mushrooms with or without stipeon the same tree trunk (Fig. 3B). The outcome of thisphenotypic plasticity on species identification bymacromorphological features is taxonomic confusion(more than 265 species have been described, manybeing invalid) (Chiu et al., unpublished result; Mon-calvo and Ryvarden, 1997). Artificial cultivation is thusa good means to determine the stability of the morpho-logical feature in this instance (Chiu et al., 2000a).Chemotaxonomy and numerical taxonomy will be abetter strategy to handle these Ganoderma systemat-ics (e. g., Sugiyama, 1998; Sugiyama et al., 1985). It isemphasized that a single gene tree cannot solve allthe problems (Berbee and Taylor, 1999; Sugiyama,1998). For instance, the internally transcribed spacerregion harbors intraspecific and interspecific variationsfor L. edodes and Pleurotus pulmonarius (Chiu et al.,

2000b; Hibbett et al., 1995, 1998) (Fig. 7). Thus a bio-logical species concept based on sexual compatibilitymust be practised to delimit species (Chiu et al.,2000a).

The population ecology of L. edodes was first re-vealed through an integrated approach of conventionaland molecular markers (Chiu et al., 1999). At present,we turn our attention to G. lucidum, a plant pathogen,and we want to understand its relationship with its restof the ecosystem. This information will enable us to as-sess the balance of fungal conservation and tree pro-tection. A better and more basic study of this type isespecially essential for the noncultivated mycorrhizalspecies, such as matsutake, for a sustainable mush-room-picking practice in the wild (Hosford et al., 1997;Molina et al., 1993; Pilz and Molina, 1996).

Fruiting is an important change from vegetativegrowth to fruiting morphogenesis (Chiu and Moore,1996, 1999a; Moore, 1998). Mating-type alleles havereceived great attention as a potential genetic control(Kües, 2000). However, with the phenomenon of hap-loid fruiting, the mating type control is at best at thechoice of partners for establishing the normal het-erokaryotic fruiting status (Leslie and Leonard, 1984).Meanwhile, we have established a simple in vitrobioassay system to examine sporulation modulatorsand fruiting modulators for Coprinus cinereus and P.pulmonarius (Chiu and Moore, 1988a, b, 1996; Chiuand To, 1993; Chiu et al., 1998a). Using fruiting bodytissues at certain physiological stages, the explantscan reinitiate the formation of fruiting bodies (a phe-nomenon called renewed fruiting) even on water agar.Heavy metals, 2-deoxy-D-glucose, and other com-pounds that act on diverse metabolic pathways can retard or inhibit renewed fruiting. Thus fruiting might be triggered by multiple pathways (Moore, 1998).Through the use of a molecular approach, varioustranscripts have been isolated from fruiting body pri-mordia and showed tissue specificity. The most thor-oughly examined class of gene products is hy-drophobins, which are potential surface-active agents(Kües, 2000; Wessels, 1999). However, most of theseidentified transcripts are the machinery for mushroommorphogenesis, e.g., rDNA amplification and mito-chondrial ATPase. The Japanese colleagues have iso-lated cerebrosides as a class of fruiting body-inducingsubstance (abbreviated as FIS), which shows cross-specificity to many mushroom species and later wasfound to show inhibitory activities on replicative DNA

2000 Mushroom exploitation in the 21st century 275

Fig. 6. The removal kinetics of pentachlorophenol (PCP)by the spent mushroom compost of Pleurotus pulmonarius in abatch culture.

An initial rapid phase of degradation within the first 2 days isachieved by immobilized enzymes in the spent mushroom com-post. The slow phase from day 2 to day 12 represents enzymeinduction of the indigenous microbes that use the nutrients inthe spent mushroom compost for growth, thus causing decay ofthe spent mushroom compost leading to a consequent drop inremoval by biosorption. The last plateau phase from day 12 today 21 refers to a fixation of PCP to the residual spent mush-room compost, leading to unavailability for biodegradation.Symbols: �, biodegradation; �, biosorption.

276 CHIU et al. Vol. 46

Fig. 7

polymerase (Mizushina et al., 1998). Thus the fungalfruiting as a developmental process is confined to aclassical cell cycle model of diverging from prolifera-tion (for cell growth that requires DNA replication) todifferentiation (for creating cell heterogeneity).

Mushrooms as a crop for consumption carry out thebiologically significant functions of meiosis, spore for-mation, and spore dispersal for species propagation.There are many ways in the architecture of a mush-room to achieve these goals (Chiu and Moore, 1996;Moore, 1998). Nevertheless, the pileus has more en-ergy and reserve materials (minerals and proteins)than the stipe, which is for mechanical support and thetranslocation of nutrients (Chiu et al., 1998a, unpub-lished result). But consumer appeal does not dependsolely on nutrition. Appearance, flavor, and taste areequally important. For instance, the dark V. volvaceamushrooms are consumed at the egg stage when theuniversal veil covers the whole immature structurecalled hymenophore. Neither the sweet flavor contentnor nutrition suggest that this human choice is wise(Chiu and To, 1993; Chiu et al., unpublished result;Mau et al., 1997)! Volvariella mushrooms autolyzewithin a few days after harvest. Cold storage will onlytrigger the senescence pathway immediately (Chen etal., 2000). Thus the harvesting of mushrooms at theegg stage will be more convenient for transportation,and the ‘eggs’ are attractive for consumption. Theshort shelf-life and low biological efficiency, however,have limited the popularity of V. volvacea in cultivationand marketing.

5. Biodiversity, Genetic Breeding, and Conservation

Biodiversity in nature is the key contribution to thegenetic resource for creating different varieties in artifi-cial breeding to suit different consumer demands. Themating-type polymorphism has traditionally been usedto reveal biodiversity in a mushroom species (Cassel-ton, 1998; Fox et al., 1994; Raper, 1966; Tokimoto etal., 1973). L. edodes usually takes 2–6 months to pro-duce mushrooms and 1–4 weeks to colonize a 9 cm

PDA plate. The conventional method of breeding in theformat of collecting sexual haploid progenies and inter-crossing them, which is followed by selection, is labor-intensive and time-consuming. Besides, a field isolatemay not produce mushrooms in laboratory settings.Thus the recovery of homokaryons by protoplasting isa comparatively easy and rapid method. Moreover,neutral DNA markers existing in every organism havebeen developed for strain authentication (Chiu et al.,1993, 1995, 1996, 2000b). A fungal protoplast/sphero-plast is a viable cytoplasm enclosed by a cell mem-brane with remnants of cell wall. Young mycelia or theimmature gills or spore germlings are good materialsfor protoplasting (Chiu et al., 1999). Protoplast technol-ogy has advanced basic studies in population genet-ics, fungal karyotype, and even sexuality for the eco-nomically important specialty mushrooms such as L.edodes and V. volvacea (Chiu and Moore, 1999b;Maeta et al., 1988; Ohmasa et al., 1987).

A biodiversity study employing protoplast technologywas carried out for L. edodes, which is native to HubeiProvince, China (Chiu et al., 2000b, unpublished re-sult). This province covers 185,900 km2 and most wildisolates differed in mating-type specificities; 50 differ-ent A-mating factors and 55 different B-mating factorswere found among 32 wild isolates. In comparison,Fox et al. (1994), employing a protoplasting technique,examined mating-type polymorphism among 17 world-wide cultivated strains and found 9 different A factorsand 10 different B factors, supporting low genetic het-erogeneity observed in most cultivars of edible mush-rooms (Chiu et al., 1993, 1996). In Japan, the 33 wildisolates collected from the field showed 41 different Afactors and 48 different B factors (Tokimoto et al.,1973). Thus higher polymorphism in mating-type fac-tors is obtained with L. edodes collected from a singleprovince in China, though a specific function or geneticstructure of the mating factors of L. edodes has notbeen determined. Furthermore, this study also reflectsthat a well-protected reserve area harbors a great pop-ulation heterozygosity for L. edodes. Protecting thenatural environment is still the most effective conser-

2000 Mushroom exploitation in the 21st century 277

Fig. 7. Nucleotide polymorphism in the ITS1 region of the wild strains of Lentinula edodes collected in China incomparison with strains of the 5 rDNA lineage groups by Hibbett et al. (1995, 1998).

The GenBank accession numbers are 5 rDNA lineage groups: Group 1, THL1, U33091 and JPN3, U33090; Group 2,PNG5, U33070 and PNG1, U33083; Group 3, NZL1, U33081 and NZL5, U33079; Group 4, PNG4, U33086 and PNG3,U33085; Group 5, NEP, AF031191. Chinese isolates: FUJ1, AF315696; HUB1, AF315697; SHA1, AF315698; SHA2,AF315694; TAI1, AF315695; YUN1, AF315693. Out-group: Pleurotus pulmonarius strain Pl27, AF315692. *, identicalbase; –, gap; N, uncertain nucleotide.

vation practice (Chiu and Moore, 2001; Chiu et al.,2000b). This is most important for China becausemore than 77% of its natural forests have been des-troyed since human inhabitation, and China claims tohave more than a million mushroom growers of thesmall-farm type scattered in mountainous areas (Lohet al., 1999). Conservation is in urgent need and im-portant. This can also be reflected by the following twoexamples: Indonesia produced 22,000 Mt mushroomsin 1995, but as a consequence of a forest fire in 1997that destroyed 150,000 ha of forest, this country pro-duced only 7,000 Mt in 1998 (http://apps.fao.org).What a great economic loss! Similarly, Tricholomamatsutake forms mycorrhizal symbiosis with Pinusdensifolia in Japan. Since a nematode attack on thetrees, the matsutake harvest in Japan has severelydropped, leading to imports from China, Morocco,North America, and Korea to meet consumer demand(Hosford et al., 1997). Protecting the natural habitat isthus necessary and essential for a sustainable mush-room industry.

When V. volvacea is analyzed, it is a unique speciesnot restricted from mating, though no regularity in mat-ing reaction is found to be used for deducing a fixedgenetic control system for sex (Chang and Yau, 1971;Chang et al., 1981; Chiu and Moore, 1999b; Royse etal., 1987). Also, there is evidence that genetic variationis observed in the wild (Chiu et al., 1995). Via proto-plast formation and regeneration, the vegetativemycelium of V. volvacea is found to be heterokaryotic(Chiu and Moore, 1999b). This genetic heterogeneitypersists in the F1 and F2 progenies of a self-fertilestrain (Chang and Li, 1991; Chang et al., 1981; Chiuand Moore, 1999b). This self-fertility together withstrain instability makes conventional breeding work dif-ficult. The greatest difficulty with the breeding work forthis mushroom species, however, is our scarce knowl-edge about its biology (Chiu, 1993). Perhaps ad-vanced and molecular biotechnological means areneeded.

In genetic manipulation, protoplasts are used in twoareas: protoplast fusion and transformation (Fincham,1989; Peberdy, 1980, 1989; Woestemeyer andWoestemeyer, 1998). Protoplast fusion is an act forhybridization to bring together whole genomes of re-lated or unrelated strains by removing the natural bar-rier of cell wall (Kevei and Peberdy, 1977; Sonnenbergand Wessels, 1987). The experimenter hopes that ge-netic recombination will follow in the hybrid (Selebano

et al., 1993; Vannacci et al., 1997; Viaud et al., 1998).However, the result of protoplast fusion may be neitherpredictable nor desirable. For instance, in the proto-plast fusion between V. volvacea and V. bombycina,the hybrid as verified by DNA fingerprints did not ex-tend the temperature limit of growth (Chiu et al., 1993).Meanwhile, the transfer of organelles/genomes by pro-toplast fusion (e.g., mitochondria, nucleus, virus) iscommonly practised (Ferenczy and Maraz, 1977; Guand Ko, 1998; Sivan et al., 1990). On the other hand,transformation is the introduction of a DNA fragment ofinterest into a cell (Allshire, 1990; Fincham, 1989;Goldman et al., 1990; Sanchez et al., 1998). Usingtransformation with a gene-encoding manganese per-oxidase from Pleurotus ostreatus, a transgenic Copri-nus cinereus strain was created, and it gained thenovel ability to break down lignin (Shishido et al.,1998). Two recent developments in the transformationsystem of mushrooms are worth attention: Restrictionenzyme-mediated DNA integration has been devel-oped to generate mutants with convenience mappingthe site of DNA insertion (Granado et al., 1997;Sanchez et al., 1998). Furthermore, adopting the sys-tem from dicotyledonous plants, the Agrobacteriumtumefaciens transmission system is found successfulin transforming A. bisporus (De Groot et al., 1998;Mikosch et al., 2000). Regardless of the technology,with the hesitation in accepting genetically modifiedfood because of its uncertainty and safety, a conven-tional breeding strategy is still the most reliable meansto generate various varieties and cultivars. Neverthe-less, conserving biodiversity is for the survival of themushroom species and serves as a resource of usefulgenes for the direct benefits and sustainability of thehuman society.

6. Concluding Remarks: Mushroom Exploitation andBasic Studies

As a manipulated group of saprotrophic or myco-rrhizal organisms, or both, mushrooms will continue tobe used for commercial exploitation. Their future usewill not be limited solely to food production. Opportuni-ties for exploitation have already been expanding intothe fields of nutraceuticals and food supplements forspecial flavor, taste, texture, and color. Advances incultivation technology guarantee the control of productquality and consistency and permit modification as re-quired to meet market demands.

278 CHIU et al. Vol. 46

Developed and developing countries grow and sellmushrooms, and the cultivation industry is currentlystructured in a way that allows many benefits to be re-turned to the world’s mushroom farmers. Exploitation,however, is an economic concept. In developing culti-vation methods, this means modernization of the tradi-tional methods and integration of mushroom cultivationwith waste disposal and remediation. The develop-ment of commercial picking means combining conser-vation with mushroom and truffle hunting so that theindustry is sustainable. In the fermentation industriesthis means ensuring that the real value of the productfar outweighs any adverse environmental effectcaused by the industrial process itself or its wastes.Above all is a need for wider and deeper research intothe biology of mushrooms. Too often it seems that liter-ature surveys reveal our ignorance more explicitly thanthey do our knowledge.

Acknowledgments

This manuscript is dedicated to Professor David Moore andProfessor Shu-Ting Chang who introduced SWC to the field ofmushroom science. SWC thanks UGC (University Grants Committee)-earmarked grants (CUHK21/91, CUHK194/94M,CUHK4077/97M), the Croucher Foundation of Hong Kong, theBritish Council, and the Chinese University of Hong Kong for re-search grants. She is sincerely grateful to Dr. W. T. Chiu, Mr. N.L. Lee, Mr. S. K. Hsui, Mr. K. L. Lau, Ms. W. M. Law, Mr. Z. M.Wang, Mr. T. M. Leung, Ms. S. Y. Ma, and Mr. P. Wang for theirtechnical assistance and collaboration. She also greatly appre-ciates her family’s support.

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