APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 3060-3066 Vol. 58, No. 90099-2240/92/093060-07$02.00/0Copyright C) 1992, American Society for Microbiology

Characteristics of a Hydrated, Alginate-Based DeliverySystem for Cultivation of the Button Mushroom

C. P. ROMAINE* AND B. SCHLAGNHAUFERDepartment of Plant Pathology, The Pennsylvania State University,

University Park, Pennsylvania 16802

Received 3 April 1992/Accepted 14 July 1992

The production of the button mushroom Agaricus bisporus with mycelium-colonized alginate pellets as aninoculant of the growing medium was investigated. Pellets having an irregular surface and porous internalstructure were prepared by complexing a mixture of 1% sodium alginate, 2 to 6% vermiculite, 2% hygramer,and various concentrations of Nutrisoy (soy protein) with calcium chloride. The porous structure allowed thepellets to be formed septically and then inoculated and colonized with the fungus following sterilization. Byusing an enzyme-linked immunosorbent assay (ELISA) to estimate fungal biomass, the matrix components ofthe pellet were found to be of no nutritive value toA. bisporus. Pellets amended with Nutrisoy at a concentrationof 0.5 to 8% supported extensive mycelial growth, as determined by significantly increased ELISA values, witha concentration of 4% being optimal and higher concentrations proving inhibitory. The addition of hydrated,mycelium-invaded pellets to the compost or casing layer supported the thorough colonization of the growingsubstrate and culminated in the formation of mushrooms that showed normal development and typicalmorphology. Yields and sizes of mushrooms were comparable from composts seeded with either colonizedpellets or cereal grain spawn. Similarly, amending the casing layer with pelletized-mycelium-colonized compostresulted in a 2- to 3-day-earlier and more-synchronous emergence of mushrooms than with untreated casing.This technology shows the greatest potential as a pathogen-free inoculant of the casing layer in the commercialcultivation of mushrooms.

The button mushroom, Agaricus bisporus (Lange) Im-bach, is grown commercially in a composted mixture ofwheat straw, manure, and other nitrogenous materials. Bio-conversion of these raw ingredients into a nutritive substratethat will support the growth and development ofA. bisporusto the exclusion of other microorganisms occurs in anelaborate composting procedure (23). In a process referredto as spawning, the prepared compost is seeded with thefungus to promote thorough mycelial invasion of the growingmedium. Because A. bisporus does not produce a spore thatcan be used to produce mushrooms with the fidelity requiredfor commercial-scale cultivation, prepared nutrient sub-strates that support mycelial growth and act as carriers totransport the fungus into the compost are utilized. Thiscomplex of a nutrient substrate and the mycelium is knownas spawn. Throughout the history of mushroom cultivation,a variety of substrates, including manure, tobacco stems,and cereal grain, have been used in the preparation of spawn(20). Today, cereal grain, either rye or millet, is usedexclusively in the mushroom industry, because of its conve-nience and reliability.Once the compost has been sufficiently colonized by the

fungus, it is overlaid with a layer of peat, which is known ascasing. Casing is a strict requirement for the formation offruiting bodies in A. bisporus, although precisely how thistriggers the transition from the vegetative state to the repro-ductive state of the fungus is unknown. As a variation ofcasing, the peat is mixed with a small proportion of A.bisporus-colonized compost, and then the mixture is appliedto the compost (16). This practice results in earlier, more-uniform formation of mushrooms and improved quality.

* Corresponding author.

Within weeks of casing, mushrooms appear and continue todevelop in a rhythmic fashion at weekly intervals.The immobilization of microorganisms in solid support

matrices, such as calcium alginate gel, has proven valuablefor the increased production of industrially important sec-ondary metabolites (2, 7, 9, 12, 19). A divergent applicationof this technology is the incorporation of mycelium intoalginate pellets for the delivery of soil-borne biocontrol fungi(8, 14, 15). The major benefits of such a delivery system area prolonged viability of the fungus in storage and an in-creased efficacy of the biocontrol agent in the field, presum-ably due to protection of the fungus by the alginate matrixfrom deleterious environmental factors. In this study, wedescribe methods for cultivating mushrooms of A. bisporusby using mycelium-invaded alginate pellets as an inoculantof the compost and casing. Unlike the conventional immo-bilization scheme, in which mycelium is entrapped in thepellets during formation and then dried, we report on atechnique for the production of texturized alginate pelletswhich are then inoculated with the fungus and disseminatedin a hydrated condition (18).

MATERIALS AND METHODS

Source and maintenance of cultures. Fungal cultures wereobtained from The Pennsylvania State University Mush-room Culture Collection. An off-white hybrid strain of A.bisporus (isolate 378), which was used throughout this study,was maintained on potato dextrose-yeast (PDY) agar at 24°Cand transferred to fresh medium every 3 to 4 weeks. Otherfungal species were grown as stationary cultures in PDYbroth at 24°C.

Preparation of alginate pellets and grain spawn. The pro-cedure for the preparation of alginate pellets was describedpreviously (18). Pellets contained 1% (wt/vol) sodium algi-

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nate (Keltone LV; Kelco Co., Clark, N.J.), 2% or 6%(wt/vol) pulverized vermiculite (W. R. Grace Co., Cam-bridge, Mass.), and for some formulations, 2% (wt/vol)hygramer (500 to 2,150 ,um; Polysystems Ltd., Glasglow,Scotland) in deionized water. Where specified, Nutrisoy(20/40-mesh defatted soy protein; Archer Daniels MidlandCo., Decatur, Ill.) was included at various concentrations asa source of nutrients. Pellets containing YPPS (4 g of yeastextract, 1 g of KH2PO4, 15 g of soluble starch, 0.5 g ofMgSO4 in 1,000 ml of water) were prepared by substitutingYPPS broth for water.For the formation of pellets, the alginate mixtures were

poured into a 12.5-cm-diameter funnel and constantly mixedwith a motor-driven chain stirrer. The mixtures were ex-truded through 2-mm-diameter tubing under gravity flow intoa 0.2 M aqueous CaCl2 solution that was being magneticallystirred. Pellets were cured for 20 min in the CaCl2 solutionand drained for 20 min in a funnel lined with Miracloth(Calbiochem Co., La Jolla, Calif.). In experiments using anenzyme-linked immunosorbent assay (ELISA) to evaluatefungal biomass, 125-ml Erlenmeyer flasks, each containing50 ml of pellets, were plugged with cotton and autoclaved for45 min. For the production of inoculant used in croppingtrials, 300 ml of pellets was contained in a 500-ml Erlen-meyer flask. After the flasks were cooled, free water thatformed was decanted. Each flask was inoculated with eithertwo 4-mm-diameter mycelial plugs or two grains of ryespawn. Flasks were placed at 24°C and shaken after 7 and 12days of growth to redistribute the inoculum. Rye or milletgrain spawn was prepared by standard procedures (20).

Preparation of anti-A. bisporus antibodies. Stationary cul-tures of A. bisporus were grown for 3 weeks in YPPS brothat 24°C. Mycelium was harvested by vacuum filtration andwashed exhaustively with distilled water. Mycelium (0.5 g)was homogenized with a Dounce homogenizer in 1.5 ml of0.05 M phosphate buffer, pH 7.0. One ml of the mycelialhomogenate was emulsified with 1 ml of Fruend's incom-plete adjuvant and administered subcutaneously at multiplesites along the back of a New Zealand White rabbit. Twomore series of injections were administered at 1-week inter-vals. One week after the final injection, the rabbit was bledby cardiac puncture. Blood was allowed to clot overnight at24°C, and the serum was clarified by centrifugation at 3,000x g for 3 min. Gamma globulins were purified by columnchromatography on CM Affi-Gel blue as described by themanufacturer (Bio-Rad Laboratories, Rockville Center,N.Y.) and conjugated to alkaline phosphatase (5).ELISA for the determination of mycelial growth on alginate

pellets. The double-antibody sandwich version of the ELISAwas carried out in microtiter plates (Costar Corp., Cam-bridge, Mass.) (5). Coating gamma globulins were used at aconcentration of 5 ,ug/ml and were incubated in the platewells at 37°C for 3 h. To prepare test samples, 1 g of pelletswas sampled from the total of 50 ml contained in a 125-mlflask and homogenized with a Brinkmann Polytron for 45 s in9 ml of PBS-T-PVP (0.02 M phosphate, 0.15 M NaCl [pH7.4] with 0.05% Tween 20 and 2% polyvinylpyrrolidone).The homogenate was diluted with 9 volumes of PBS-T-PVP,vortexed, and incubated in duplicate in plate wells at 6°C for16 to 18 h. In experiments to test the specificity of theantibodies, 0.1 g of mycelium or mushroom tissue of a fungalspecies was homogenized with 10 ml of PBS-T-PVP, clari-fied by centrifugation at 14,000 x g for 1 min, and incubatedin the plate wells as described above. For all experiments,phosphatase-conjugated gamma globulins were diluted 250-fold and incubated at 37°C for 3 h. To assess the antibody

reaction, a 1-mg/ml solution ofp-nitrophenyl phosphate wasincubated in the well at 240C for 15 to 30 min, and thenitrophenolate end product was quantified by measuringA405. Under these assay conditions, color development waslinear with respect to the dilution of the homogenate and thelength of the substrate reaction. ELISA values (A405) werecalculated by subtracting the mean absorbance value for aPBS-T-PVP buffer control from the mean observed absor-bance value for the treatment. Experiments were conductedtwice with two or three replications per treatment perexperiment. Statistical analysis was performed according tothe Waller-Duncan K-ratio t test or Fisher's least significantdifference (24).

Scanning electron microscopy. Alginate pellets were fixed,dehydrated, cryofractured, and dried at the critical point(10). Specimens were coated with 28 nm of gold and viewedwith a scanning electron microscope (model 60; Interna-tional Scientific Instruments, Pleasanton, Calif.). Photo-graphic records of fractured surfaces were taken with Po-laroid type 52 film. Montages of photomicrographs taken ata magnification of x400 were constructed to document thesite and extent of mycelial colonization.Mushroom production trials. Cropping trials were carried

out essentially as outlined by Carroll and Schisler (3).Compost was prepared according to the short method (23) byusing wheat straw-bedded horse manure supplemented withbrewer's grain, poultry manure, and gypsum at the rate of50, 28, and 50 kg per dry metric ton of horse manure,respectively. For spawning, 110 g of rye grain spawn oralginate pellets was mixed with 23 kg of compost and placedin a tray measuring 14 by 60 by 60 cm. Where indicated, eachtray of compost was supplemented at spawning with 454 g ofSpawn Mate II SE (Spawn Mate Co., Capitola, Calif.). Thecompost was maintained at 24°C for 13 days for mycelialcolonization and then cased with a 3.5-cm-thick layer ofpeat. For the production trials involving inoculants of thecasing, 182 g of either colonized alginate pellets, colonizedshredded compost, or millet grain spawn was mixed with 5.5kg of peat and applied to the surface of the compost in a tray.The temperature was maintained at 24°C in the compost for6 to 9 days, at which time it was lowered to 18°C to inducethe formation of mushrooms. Throughout the cropping cy-cle, the air temperature was maintained at 18°C, and thecasing was routinely watered to field capacity. In an exper-iment, four or five replicate' trays for each treatment werearranged in an incomplete randomized block design withinthe growing room. The weight and number of mushroomsharvested from each tray were recorded daily for 21 or 28days. Statistical analysis of the data was done by using theWaller-Duncan K-ratio t test (24).

RESULTS

ELISA for the determination of mycelial growth on alginatepellets. Polyclonal antibodies prepared against total mycelialantigens ofA. bisporus gave a comparably strong reaction inthe ELISA (A405, >2.15) with extracts of both mycelium andfruiting body tissue (P = 0.05) (Table 1). Significantly lowerELISA values (A405, <1.79) were obtained with a closelyrelated mushroom species, Agaricus bitorquis, and themushroom species Lentinula edodes (P = 0.05). In contrast,no cross-reactivity was detected for 18 other fungal species,including two mushroom fungi, Pleurotus ostreatus andFlammulina velutipes (P = 0.05). A linear relationship (r2 =0.98) existed between the ELISA absorbance value and thefresh weight of mycelium over the range of 1 to at least 100

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TABLE 1. Specificity of the polyclonal antibodies to mycelialantigens of A. bisporusa

ELISA valueTest antigen (405)b

Agaricus bisponrs (mushroom) ........................ 2.23 AAgaricus bisporus ........................ 2.16 AAgaricus bitorquis........................ 1.78 BLentinula edodes (mushroom) ............... ......... 0.77 CLentinula edodes ........................ 0.61 CDPleurotus ostreatus (mushroom) ........................ 0.17 DEPleurotus ostreatus ...... .................. 0.13 EAspergillus fumigatus ........................ 0.07 EMycogone sp......................... 0.07 ETnchoderma viride........................ 0.05 EFlammulina velutipes........................ 0.02 EChaetomium olivaceum........................ 0.02 EChrysosporium sp......................... 0.01 EBotryotrichum sp......................... 0.00 EDactylium dendroides ........................ 0.00 EDiehliomyces sp......................... 0.00 EOedocephalum sp......................... 0.00 EOidiodendron sp......................... 0.00 EPapulaspora byssina........................ 0.00 EPenicillium sp......................... 0.00 ESependonium sp......................... 0.00 ESporotrichum sp......................... 0.00 ETnchothecium sp......................... 0.00 EVerticillium fungicola ........................ 0.00 E

a Mycelium of each fungal species was grown in stationary liquid culture,harvested by vacuum filtration, and tested by the ELISA with polyclonalantibodies to mycelial antigens of A. bisporus. Where indicated, mushroomtissue was substituted for mycelium as the test sample.

b ELISA values represent the means of a total of four replications in twoexperiments. Means followed by the same letter are not significantly different,according to the Waller-Duncan K-ratio t test at P = 0.05.

jig (data not shown). A significantly positive ELISA valuecompared with the buffer control value was obtained withmycelial antigens representing as little as 2 ,ug (fresh weight)of tissue.By using the ELISA to quantify mycelial growth, the

structural components of the pellets, specifically 1% algi-nate, 6% vermiculite, and 2% hygramer, were found to be ofno nutritional value to A. bisporus (P = 0.05) (Table 2).However, the addition of YPPS broth or Nutrisoy to theformulation resulted in appreciable vegetative growth. Theaccumulation of fungal biomass was comparable in pelletscontaining either YPPS broth or 1% Nutrisoy (P = 0.05). Asignificant increase in mycelial growth resulted when theconcentration of Nutrisoy was increased from 1 to 4% (P =0.05). In experiments designed to determine the optimalconcentration of Nutrisoy, fungal growth increased linearly

TABLE 2. Mycelial growth of A. bisporus on differentformulations of alginate pelletsa

Pellet composition ELISA value( 405)b

Matrix (1% alginate-6% vermiculite-2% hygramer) ..... 0.00 CMatrix + YPPS broth ............................................ 0.36 BMatrix + 1% Nutrisoy ........................................... 0.29 BMatrix + 4% Nutrisoy ........................................... 0.65 A

a Alginate pellets were inoculated with A. bisporus and assayed by theELISA for fungal biomass at 18 days postinoculation.

b ELISA values represent the means of a total of six replications in twoexperiments. Means followed by the same letter are not significantly differentaccording to the Waller-Duncan K-ratio t test at P = 0.05.

0.8

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% NutrisoyFIG. 1. Relationship between the concentration of Nutrisoy in

the alginate pellets and the accumulation of mycelial biomass of A.bisporus. Mycelial growth was quantified by the ELISA and isexpressed as A405. Fisher's least significant difference (L.S.D.) is0.05 A405 units at P = 0.0001.

over the range of 0.5 to 4%, with 4% being optimal andhigher levels proving inhibitory (P = 0.0001) (Fig. 1). Similardose-response curves were obtained with other sources ofnutrients, such as brewer's grain and the commerciallyavailable mushroom supplements Spawn Mate II SE andFast Break (data not shown).

Nutrisoy-fortified alginate pellets containing vermiculiteand hygramer typically had an ovoid shape, measuring 3 to 4by 5 to 7 mm. After inoculation, pellets developed profusemycelial growth on the surface and, when completely colo-nized, had an appearance that was similar to that of rye grainspawn. After the pellets were shaken to redistribute theinoculum, visible regrowth of mycelium on the pellets wasordinarily observed within 18 to 22 h. Scanning electronmicroscopic analysis revealed that mycelium had ramified anextensive network of internal cavities through rifts in thesurface of the pellets (Fig. 2).

Alginate pellets as an inoculant of the compost. In twomushroom production trials, A. bisporus-colonized alginatepellets were substituted for conventional rye grain spawn asan inoculant of the compost. In both trials, the extent ofmycelial colonization of the compost at 13 days after spawn-ing, the time when casing is applied, was similar for grainspawn and alginate pellets containing 4% Nutrisoy. Theyields of mushrooms from unsupplemented compostspawned with grain spawn and alginate pellets containing 4%Nutrisoy were statistically comparable (P = 0.05) (Table 3).A slightly weaker colonization of the compost and a loweryield were associated with alginate pellets containing 6%Nutrisoy (P = 0.05). This trend was observed in bothexperiments, although yields were higher in the secondexperiment because of the addition of the delayed-releasesupplement Spawn Mate II SE to the compost (Table 3). Nosignificant difference in the sizes of mushrooms existedamong the treatments (P = 0.05). Mushrooms grown fromalginate pellets showed normal development and morphol-ogy (Fig. 3, treatments 2 and 3), being indistinguishable fromthose arising from grain spawn (Fig. 3, treatment 1).

Alginate pellets as an inoculant of the casing. Seeding the

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FIG. 2. Scanning electron microscopy of a freeze-fractured A. bisporus-colonized alginate pellet. Pellets contained 1% alginate, 2%vermiculite, 2% hygramer, and 4% Nutrisoy. Mycelium is indicated by the arrows. Bar represents 1 mm.

casing with A. bisporus-colonized compost or pellets re-sulted in a more-rapid growth of the fungus through thecasing layer and a 2- to 3-day-earlier appearance of mush-rooms than with unamended casing (Fig. 4). Also, bothpellets and compost promoted a more-uniform developmentof mushrooms, as evidenced by the shortened harvest periodrequired for each rhythmic flush (Fig. 5). Whereas approxi-mately 4 days were required to harvest the mushrooms ateach flush for unamended casing, only 2 to 3 days were

TABLE 3. Production of mushrooms in compost spawnedwith alginate pelletsa

Mushroom productionb

Spawning agent Expt 1 Expt 2

Yield Size (g) Yield Size (g)(kg.m2) Sz g (kg.-m-2)

Rye grain 12.70 AB 7.30 A 15.44 A 7.20 AAlginate pelletsc + 10.85 BC 7.90 A 13.68 AB 7.38 A4% Nutrisoy

Alginate pelletsc + 10.75 C 7.41 A 13.33 B 7.19 A6% Nutrisoya A. bisporus-colonized alginate pellets or rye grain was used to spawn

compost. The compost was not supplemented in experiment 1 and wassupplemented with Spawn Mate II SE in experiment 2.

b Determined after 21 days of production. Data represent the mean valuesof five replications. Means within a column followed by the same letter are notsignificantly different according to Waller-Duncan K-ratio t test at P = 0.05.

C 1% alginate and 6% vermiculite.

needed when the casing was amended with colonized pelletsor compost. Moreover, there was a tendency for mushroomsto develop singly rather than in clumps, as was often thesituation with unamended casing. After 28 days of produc-tion, no significant difference in the yields, morphology, andsizes of mushrooms existed among the treatments, with theexception of millet grain spawn, which was associated witha yield loss and larger mushrooms (P = 0.05) (Table 4).Millet grain, although supporting vigorous mycelial growthin the casing layer, failed to induce the earlier and more-synchronous maturation of mushrooms.

DISCUSSION

A synthetic substrate-based delivery system for the culti-vation of A. bisporus would allow for greater flexibility andcontrol of those factors which impact vegetative growth andfruiting than is possible with existing substrates. Here, wehave described a method whereby mycelium-colonized, nu-trient-fortified calcium alginate pellets can be used to seedthe growing medium for the production of mushrooms. Inthis regard, the alginate pellets functioned as an analog ofgrain spawn and shredded colonized compost, which areemployed commercially as inoculants of the compost andcasing layer, respectively. Moreover, with alginate pellets asan inoculant, mycelial colonization of the growing mediumoccurred in a typical fashion and the mushrooms that formeddeveloped normally and showed characteristic size andmorphology.

In the traditional immobilization procedure, the organism

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FIG. 3. Production of mushrooms in compost inoculated withrye grain spawn (treatment 1), A. bisporus-colonized alginate pelletswith 4% Nutrisoy (treatment 2), and A. bisporus-colonized pelletswith 6% Nutrisoy (treatment 3). The third flush of mushrooms isshown.

is grown in liquid culture axenically and then entrapped inpellets as droplets of a mixture of sodium alginate andculture broth contact a calcium solution (2). In this study, wehave produced texturized pellets having an irregular surfaceand porous internal structure by incorporating vermiculiteand hygramer into the alginate formulation. Following theirformation, these pellets could be sterilized and inoculatedwith A. bisporus, in much the same manner as grain spawnis prepared commercially (20). Microscopic analysis re-vealed that the fungus invaded interior regions of the pelletsby growing through openings in the surface created by thetexturizing agents. In addition to the modified immobiliza-tion scheme, we inoculated the mushroom-growing mediumwith hydrated pellets instead of conventional dried pellets.We reasoned that in a hydrated form, the fungus wouldbecome more rapidly established in the compost and casing,although we have not evaluated the efficacy of dried pellets.Such a study should consider the use of an osmoregulantsuch as polyethylene glycol in the drying process, since thiscompound has been shown to enhance the mycelial growthor sporulation of the biocontrol fungi Tichoderma har-zianum and Beauveria bassiana in alginate pellets (14).

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FIG. 4. Production of mushrooms with casing that was un-amended or amended with A. bisporus-colonized compost (com-post), millet grain spawn (millet spawn), or A. bisporus-colonizedpellets with 1% alginate, 6% vermiculite, 2% hygramer, and 4%Nutrisoy (alginate pellets). Shown is the emerging first flush ofmushrooms at 15 days postcasing.

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FIG. 5. Time course for the development of mushrooms asinfluenced by inoculants of the casing layer. The casing was un-amended (treatment [Trt] 1) or amended with A. bisponrs-colonizedcompost (Trt 2), millet grain spawn (Trt 3), orA. bisporus-colonizedpellets with 1% alginate, 6% vermiculite, 2% hygramer, and 4%Nutrisoy (Trt 6). Shown is the day-to-day harvest for the first fourflushes of mushrooms.

We used anti-A. bisporus polyclonal antibodies in anELISA format as a quantitative assay for fungal biomass.For us, the ELISA was a convenient, expeditious, andhighly reproducible test that proved invaluable in optimizingnutrient formulations of pellets for maximal mycelial growth.Our data suggest that polyclonal antibodies to total mycelialantigens are reasonably specific for A. bisporus, cross-reacting with only two other mushroom species among

TABLE 4. Effect of amending the casing with A. bisponus-colonized alginate pellets on the production of mushroomsa

Mushroom productionbCasing amendment Yield

(kg_ M2) Size (g)

None 8.74 A 7.51 BCompost 8.69 A 5.39 BMillet grain 5.86 B 9.94 AAlginate pelletsc + 4% Nutrisoy 8.89 A 6.48 BAlginate pelletsc + 8% Nutrisoy 9.08 A 6.47 BAlginate pelletsd + 4% Nutrisoy 8.65 A 5.37 B

a A. bisponus-colonized compost, millet grain, or alginate pellets weremixed with casing and overlaid on spawn-grown compost.

b Determined after 28 days of production. Data represent the mean valuesof two experiments with four replications per experiment. Means within acolumn followed by the same letter are not significantly different according toWaller-Duncan K-ratio t test at P = 0.05.

c 1% alginate and 2% vermiculite.d 1% alginate, 6% vermiculite, and 2% hygramer.

twenty phylogenetically diverse fungi. The production ofpolyclonal and monoclonal antibodies to a wide variety offungal species has been described previously (1, 4, 6, 11, 13,17, 21, 26) and suggests that the ELISA procedure describedherein for estimating biomass of A. bisporus should begenerally applicable to fungi. In fact, there are severalexamples in which seroassays have been adapted for thequantitative analysis of fungi (4, 13, 17, 21). For quantifica-tion, a polyclonal antibody-based ELISA would be preferredto a monoclonal antibody test, because absorbance would bea measure of a large number of epitopes, rather than a singleone, and consequently, would not be sensitive to fluctua-tions in the level of epitopes occurring independently ofmycelial growth.A salient feature of the substrate used for spawn is that it

contains a nutrient reserve which supports the rapid andthorough colonization of the compost byA. bisporus; other-wise, competing fungi can flourish and reduce the yield ofmushrooms (22, 25). We have found that alginate pelletscontaining 4% soy protein (Nutrisoy) promoted a degree ofcolonization of the compost comparable to that of conven-tional grain spawn. Further, a similar yield of mushroomswas obtained from compost spawned with either of theseinoculants. The lower productivity associated with 6% Nu-trisoy is probably related to the more-limited mycelial inva-sion of the compost that was noted. We have observed thata low-nutrient status of the pellet produced a more-invasivegrowth, whereas higher levels supported a dense mycelialgrowth that had a tendency to remain confined to the pelletrather than growing into the compost. In our opinion,formulations of pellets that improve the rate of colonizationof the compost or increase the yield and quality of mush-rooms in comparison to grain spawn must be identifiedbefore the use of this technology will become cost-effective.

Inoculating the casing layer with pelletized myceliumhastened both the colonization of the casing and the subse-quent development of mushrooms. Also, mushroomsemerged more uniformly, and quality was improved becauseof a diminished clumping of the developing fruiting bodies.The response observed with alginate pellets closely paral-leled that obtained with the addition of shredded colonizedcompost (16). The earlier and more-synchronous appearanceof mushrooms results from the mycelium being uniformlydispersed throughout the casing, thereby allowing it to growevenly to the surface, regardless of the depth of the casing.With unamended casing, mycelium must traverse the casinglayer from the underlying compost to reach the surface, andin doing so, irregularities in the thickness of the casing createuneven surface growth and a delayed and asynchronousformation of mushroom primordia. However, a faster colo-nization of the casing layer does not fully explain the alteredpattern of fruiting obtained with inoculants. Millet grainspawn, although stimulating mycelial growth, did not evokethe early development of mushrooms. This would suggestthat vegetative growth and the initiation of primordia aredistinct processes, with perhaps the latter being more sensi-tive to some feature of the inoculant (for example, the natureof the nutrient reserve). Also, in contrast to compost inwhich a higher nutrient status of the pellet diminishedcolonization, this was not a factor in the casing layer.Inoculation of the casing with pellets containing 4 and 8%Nutrisoy yielded a similar response. In fact, amending thecasing with high levels of nutrients can, under certaincircumstances, significantly increase yield (18a). The appar-ent discrepancy in the importance of the nutrient level on thecolonization of compost and casing is unclear but might

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involve the large disparity in nutrient status that existsbetween these two substrates.

Presently, the technology described here shows greatestcommercial promise as an inoculant of the casing layer.Clearly, seeding the casing with shredded, colonized com-post is advantageous with regard to improved quality andease of harvesting of mushrooms, but it is practiced to onlya limited extent commercially because of the disease threatposed by the widespread dispersal of a potentially pathogen-infested substrate. An alginate-based delivery system wouldoffer a pathogen-free mechanism for the large-scale introduc-tion of the fungus into the casing. Additionally, this technol-ogy might be used to research the fruiting process of A.bisporus as influenced by factors within the casing layer aswell as to potentially deliver bioactive materials to the casingthat increase the yield and quality of mushrooms or regulatethe development of mushrooms. Broader application ofhydrated, texturized alginate pellets might extend to a car-rier for the dissemination of biocontrol fungi that are recal-citrant to drying and as a solid support matrix for fermenta-tive fungi, whereby the fungus could be introduced into thebioreactor and the matrix could be colonized in situ.

ACKNOWLEDGMENTS

This work was supported by Pennsylvania Department of Agri-culture grant no. ME449010.We gratefully acknowledge the technical assistance of Roxanne

Davis, Rosemary Walsh, and Harry Muthersbaugh.

REFERENCES1. Aidwell, F. E. B., I. R. Hall, and J. M. B. Smith. 1983.

Enzyme-linked immunosorbent assay (ELISA) to identify en-domycorrhizal fungi. Soil Biol. Biochem. 15:377-378.

2. Brodelius, P., and K. Mosbach (ed.). 1987. Immobilizationtechniques for cells/organelles. Methods Enzymol. 135:171-189.

3. Carroll, A. D., and L. C. Schisler. 1976. A delayed releasenutrient for mushroom culture. Appl. Environ. Microbiol. 31:499-503.

4. Casper, R., and K. Mendgen. 1979. Quantitative serologicalestimation of a hyperparasite: detection of Verticilfium lecanii inyellow rust infected wheat leaves by ELISA. Phytopathol. Z94:89-91.

5. Clark, M. F., and A. N. Adams. 1977. Characteristics of themicro-plate method of enzyme-linked immunosorbent assay forthe detection of plant viruses. J. Gen. Virol. 34:475-483.

6. Dewey, F. M., and C. M. Brasier. 1988. Development of ELISAfor Ophiostoma ulmi using antigen-coated wells. Plant Pathol.37:28-35.

7. Figueiredo, Z. M. B., and L. B. Carvalho, Jr. 1991. L-Malic acidproduction using immobilized Saccharomyces cerevisiae. Appl.Biochem. Biotechnol. 30:217-224.

8. Fravel, D. R., J. J. Marois, R. D. Luimsden, and W. J. Connick,Jr. 1985. Encapsulation of potential biocontrol agents in analginate-clay matrix. Phytopathology 75:774-777.

9. Galazzo, J. L., and J. E. Bailey. 1990. Growing Saccharomycescerevisiae in calcium alginate beads induces cell alterations

which accelerate glucose conversion to ethanol. Biotechnol.Bioeng. 36:417-426.

10. Humphreys, W. J., B. 0. Spurlock, and J. S. Johnson. 1974.Critical point drying of ethanol-infiltrated, cryofractured biolog-ical specimens for scanning electron microscopy, p. 275-282. In0. Johari and I. Korvin (ed.), Scanning electron microscopy II.TRI, Chicago.

11. Johnson, M. C., T. P. Pirone, M. R. Siegel, and D. R. Varney.1982. Detection of Epichloe typhina in tall fescue by means ofenzyme-linked immunosorbent assay. Phytopathology 72:647-650.

12. Kautola, H., W. Rymowicz, Y. Linko, and P. Linko. 1991.Production of citric acid with immobilized Yarrowia lipolytica.Appl. Microbiol. Biotechnol. 35:447-449.

13. Kitagawa, T., Y. Sakamoto, K. Furumi, and H. Ogura. 1989.Novel enzyme immunoassays for specific detection ofFusaniumoxysporum f. sp. cucumerinum and for general detection ofvarious Fusarium species. Phytopathology 79:162-165.

14. Knudsen, G. R., D. J. Eschen, L. M. Dandurand, and Z. G.Wang. 1991. Method to enhance growth and sporulation ofpelletized biocontrol fungi. Appi. Environ. Microbiol. 57:2864-2867.

15. Lewis, J. A., and G. C. Papavizas. 1987. Application of Tricho-derma and Gliocladium in alginate pellets for control of Rhizoc-tonia damping off. Plant Pathol. (London) 36:438-446.

16. MacCanna, C., and J. B. Flanagan. 1972. Casing types andtechniques. Mushroom Sci. 8:727-731.

17. Mendgen, K. 1986. Quantitative serological estimations of fun-gal colonization, p. 50-59. In N. J. Fokkema and J. Van DenHeuvel (ed.), Microbiology of the phyllosphere. CambridgeUniversity Press, Cambridge.

18. Romaine, C. P., C. E. Nelson, and R. Davis. February 1989.Synthetic substrate for filamentous fungi. U.S. patent 4,803,800.

18a.Romaine, C. P., and B. Schlagnhaufer. Unpublished results.19. Roukas, T., H. Lazarides, and P. Kotzekidou. 1991. Ethanol

production from deproteinized whey by Saccharomyces cerevi-siae cells entrapped in different immobilization matrices. Milch-wissenschaft 46:438-441.

20. San Antonio, J. P. 1984. Origin and improvement of spawn ofthe cultivated mushroom Agaricus brunnescens. Hortic. Rev.6:85-115.

21. Savage, S. D., and M. A. Sall. 1981. Radioimmunosorbent assayfor Botrytis cinerea. Phytopathology 71:411-415.

22. Schisler, L. C. 1982. Biological and mycological aspects ofmushroom composting, p. 3-10. In P. J. Wuest and G. D.Bengston (ed.), Penn State handbook for commercial mushroomgrowers. Pennsylvania State University, University Park, Pa.

23. Sinden, J. W., and E. Hauser. 1950. The short method ofcomposting. Mushroom Sci. 1:52-59.

24. Steel, R. G. D., and J. H. Torrie. 1980. Multiple comparisons, p.172-194. In Principles and procedures of statistics: a biometricalapproach, 2nd ed. McGraw-Hill, New York.

25. van Gils, J. J. 1988. Cultivation, p. 263-308. In L. J. L. D. vanGriensven (ed.), The cultivation of mushrooms. Somycel S. A.,Langeais, France.

26. Wright, S. F., J. B. Morton, and J. E. Sworobuk. 1987. Identi-fication of a vesicular-arbuscular mycorrhizal fungus by usingmonoclonal antibodies in an enzyme-linked immunosorbentassay. Appl. Environ. Microbiol. 53:2222-2225.

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