Vol. 45, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p. 511-5150099-2240/83/020511-05$02.00/0Copyright 0 1983, American Society for Microbiology

Biological Control of Olive Green Mold in Agaricus bisporusCultivation

T. E. TAUTORUS* AND P. M. TOWNSLEY

Department ofFood Science, University ofBritish Columbia, Vancouver, British Columbia, Canada V6T 2A2

Received 3 September 1982/Accepted 4 November 1982

Successful methods to control the damaging weed mold Chaetomium olivaceum(olive green mold) in mushroom beds are not presently known. An attempt was

made to control C. olivaceum by biological means. A thermophilic Bacillus sp.

which showed dramatic activity against C. olivaceum on Trypticase soy agar

(BBL Microbiology Systems)-0.4% yeast extract agar plates was isolated fromcommercial mushroom compost (phase I). When inoculated into conventional andhydroponic mushroom beds, the bacillus not only provided a significant degree ofprotection from C. olivaceum, but also increased yields of Agaricus bisporus.

The commercial cultivation of the whitemushroom, Agaricus bisporus Lange (Agaricusbrunnescens), continues to rank third overall ineconomic importance as a vegetable crop inCanada (1). Although traditionally accepted as ahorticultural craft, mushroom production is inessence an industrial fermentation process (10,13). Presently, mushroom culture represents theonly major process in biotechnology which suc-cessfully converts cellulosics into useful foodsand by-products. The methods used in this solid-state fermentation have not changed significant-ly over the years. Because of the current natureof the growing conditions (i.e. use of animalmanures, plant materials, chemical fertilizers,and other agricultural residues as a substrate),the cultivation of the mushroom is susceptible tomany competitive organisms and weed fungi (6,7), causing substantial loss of dollars to thegrower due to decreased yields (15). One suchorganism is Chaetomium olivaceum, more com-monly known as olive green mold. Olive greenmold is a weed mold which frequently occursafter pasteurization (phase II) of the compostand severely inhibits mushroom mycelial devel-opment by competing for nutrients (3, 8). Conse-quently, spawn growth is retarded or fails alto-gether. The growth of C. olivaceum inmushroom beds is very rapid and has beenknown to spread onto sterilized soil andthroughout wooden shelving (2). One reason forthe invasion of C. olivaceum into mushroombeds is the presence of free ammonia in thecompost after the phase II pasteurization (4, 17).The presence of free ammonia in compost, al-though toxic to the mushroom mycelia, resultsin production of compounds which readily sup-port growth of olive green mold (19). As yet,

there are no known methods to successfullycontrol this pest (22).From earlier research (11, 16, 20, 21) it was

suggested that a certain degree of protectionfrom invasion of the mushroom compost bydisease-causing organisms may be obtained byprior fermentation of the compost with selectedmicroorganisms. Huhnke (11) states that, byinoculating specific thermophilic microorga-nisms into sterilized compost (i.e. Till sub-strate), the cultures are capable of causing aselective protection of the substrate against dis-eases and pests. Hence, the objectives of thisinvestigation were to select thermophilic micro-organisms which would not only support themushroom, but also protect it from damage byC. olivaceum. Furthermore, it was hoped thatthis thermophilic organism could be used in thepreparation of a hydroponic system utilizingsynthetic substrates (thereby eliminating the po-tential hazards of compost) as a growth mediumfor A. bisporus. If successful, the results of thisresearch should facilitate the emergence of themushroom industry into a level of technologyexperienced in modem industrial fermentations.

MATERIALS AND METHODS

Selection of thermophiles for activity against C. oliva-ceum. Representative (45-g) samples of commercialcompost which had been passed through the initialstandard mushroom composting stage (phase I) beforedelivery to the grower for pasteurization (phase II)were received on a weekly basis from the FraserValley Mushroom Growers Association. Upon re-ceipt, these samples were immediately incubated for 2days at 55°C under aerobic conditions and high humid-ity (Fisher Isotemp incubator). This incubation provid-ed a controlled duplication of the final thermophilic

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512 TAUTORUS AND TOWNSLEY

treatment given by the mushroom grower before in-oculation which mushroom spawn.For bacteriological analysis, 10 g of the thermophilic

compost was mixed with 90 ml of sterile distilledwater, and the sample was kneaded thoroughly toplace sufficient microflora into suspension. Thereaf-ter, 10-fold dilutions, using sterile distilled water, wereprepared to a l0' dilution. Samples of 0.1 ml from the10-2 to 10-7 dilutions were spread plated in duplicateon TSY agar (Trypticase soy agar [BBL MicrobiologySystems] + 0.4% yeast extract [Difco Laboratories])and incubated at 55°C for 16 to 20 h. After incubationand the appearance of colonies, all plates weresprayed with a spore suspension of C. olivaceum. Thetreated plates were then incubated at 25°C and exam-ined daily for the presence of zones of fungal inhibi-tion.Use of Bacilus AOG (see below) in conventional

mushroom production. A 500-g portion of standardphase II mushroom compost was spawned with 9.0 gof A. bisporus spawn grains (obtained from FraserValley Mushroom Growers Association) and placed inplastic growing trays (8 by 15 by 23 cm). This spawnedmushroom compost was then subjected to variousexperimental treatments: (i) control-no further addi-tion to the compost bed; (ii) 100 ml of a 105/mlthermophilic Bacillus AOG culture grown in TSYbroth for 96 h at 55°C (New Brunswick Psychrothermshaker) was added; (iii) 100 ml of a 105/ml thermophilicBacillus AOG culture grown in TSY broth for % h at55°C was added, followed by spraying 2 ml of a C.olivaceum spore suspension on top of the bed; (iv) 2 mlof a C. olivaceum spore suspension was sprayed ontop of the bed only. All experiments were conducted in

triplicate (except iv, which was done in duplicate).The growing trays were then incubated at 25°C for

12 days to promote spawn growth. During this incuba-tion, the mycelial diameters of the spawn in each traywere measured on a daily basis (for 7 days) as anindication of the mycelial development under thevarious treatments (5). After 12 days, each of the trayswas cased with 2.5 cm of a sterile peat-sand-groundlimestone (dolomite) mixture in a ratio of 5:5:1 andplaced in a 16°C incubator to promote pinhead forma-tion. Air was pumped in at 550 ml/min to keep CO2levels at a minimum. Relative humidity was main-tained at 80%. As the mushroom fruiting bodies ap-peared (harvested just before opening of gills), yieldsfrom each tray were determined over a 1-month peri-od.Use of Bacillus AOG in hydroponic mushroom pro-

duction. A 2% (wt/vol) malt extract (Difco) solutioncontaining 2% CaCO3 and 0.4% yeast extract (pH 7.0)or a liquid compost solution was chosen as the liquidnutrient for mushroom hydroponic culture. The liquidcompost was prepared by adding 5 liters of distilledwater to 1 kg (wet weight) of mushroom compost. Themixture was agitated vigorously, and extract wascollected by filtration through cheesecloth. The ex-tract was then sterilized at 15 lb/in2 for 15 min and wascalled liquid compost (final pH, 7.2). The BacillusAOG culture was inoculated into this liquid compostand into the 2% malt extract. These inoculated liquidnutrients were then incubated at 55°C for 96 h to yielda final concentration of 104 cells per ml. The experi-mental treatments were similar to the solid compostplan previously described. A 500-ml amount of a104/ml Bacillus AOG culture in either liquid compost

FIG. 1. Inhibition of olive green mold by the isolated Bacillus sp.

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BIOLOGICAL CONTROL OF C. OLIVACEUM

2.5+

I-

a

.1

4

Ii

2.0+

1.5±

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FIG. 2. Rate of mycelial development in standardmushroom compost. Symbols: *, control; 0, Bacillussp. added; 0, C. olivaceum added; O, C. olivaceumand Bacillus sp. added.

or 2% malt extract was added to 180 g of sterilevermiculite (inert carrier material, no less than 2.0 mmin size) within a plastic tray. A sterile glass-wool plugapproximately 20 cm long was placed on the bottom ofthe tray to ensure aerobic conditions within the bed.After the spawning, representative trays with or with-out the bacillus were sprayed with 2 ml of a C.olivaceum spore suspension and incubated at 25°C for12 days. All trays were then cased with 2.5 cm of asterile peat-sand-ground limestone mixture and main-tained at 16°C for fruiting body formation. Yields ofmushrooms were recorded during a 30-day croppingperiod. All hydroponic experiments were done intriplicate.

RESULTS AND DISCUSSIONAfter approximately 4 months of phase I com-

post examinations, a thermophilic Bacillus sp.

2.5+

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FIG. 3. Rate of mycelial development in 2% maltextract. Symbols: U, control; 0, Bacillus sp. added;0, C. olivaceum added; l, C. olivaceum and Bacillussp. added.

was isolated which showed dramatic activityagainst C. olivaceum on TSY agar plates (Fig.1). The Bacillus sp. (Bacillus AOG ["anti-olivegreen"]) isolated was a facultatively anaerobic,sporeforming, gram-positive rod, motile, cata-lase positive, Voges-Proskauer positive, and ca-pable of growth in 10% NaCl and at pH 5.7. Itresembles the thermophile Bacillus coagulans inthat it was resistant to 0.02% sodium azide whengrown at 55°C and was acidophilic (9). Theisolation procedure indicated that Bacillus AOGwas capable of producing a potent antibioticagainst C. olivaceum (as shown on TSY agar).The chemical identity of this bacillus-producedinhibition is now under study.To determine whether this thermophile would

support growth of the mushroom, as well as

TABLE 1. Yield of A. bisporus fruitbodies

Yield (g, fresh wt) with given substratea

Treatment HydroponicConventional

2% malt extract Liquid compost

Control 172.1 1.5 9.0Bacillus sp. added 171.0 3.1 10.5C. olivaceum only 73.8b 0.0 0.0Bacillus sp. + C. olivaceum 138.0 3.9 16.5

a Average of three trays.b Average of two trays.

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514 TAUTORUS AND TOWNSLEY

FIG. 4. Poor spawn growth in hydroponic trays with only olive green mold present (a) and improved mycelialdevelopment due to the protection of the bacillus against olive green mold (b).

protect it from damage by olive green mold,Bacillus AOG was inoculated into two kinds ofculture medium. One medium was conventional,consisting of standard phase II mushroom com-post, and the other was hydroponic, consistingof liquid substrates absorbed onto an inert physi-cal support, vermiculite.

Figure 2 represents the rate of mushroommycelial development in standard compost overa 7-day period. When the bacillus was added tothe compost, the rate of mycelial development inthe trays was enhanced. In addition, the bacillusexhibited a definite inhibitory effect on the de-velopment of olive green mold (F test on slopes,P < 0.01). This biological protection was furtherindicated (Table 1) by a significant 86.5% in-crease in mushroom yield of the trays containingbacillus and olive green mold over that of thetrays containing olive green mold only (analysisof variance, P < 0.05). Furthermore, trays withonly C. olivaceum produced fruiting bodies a fullweek later than those with Bacillus AOG and C.olivaceum together.When the bacillus was added to hydroponic

trays in 2% malt extract (Fig. 3), a remarkableimprovement in the rate of mycelial develop-ment occurred (P < 0.01). The improved rate ofmushroom mycelial growth is shown more vivid-ly in photographs of the hydroponic trays (Fig.4). Figure 4a represents a tray with only C.olivaceum added; relatively poor spawn growthis evident. Figure 4b represents the effect of theBacillus AOG and olive green mold present at

the same time. As these results show, the mush-room mycelia flourished in the presence of thebacillus.

2.5

a

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D AYSFIG. 5. Rate of mycelial development in liquid

compost. Symbols: U, control (sterile); 0, control(nonsterile); 0, Bacillus sp. added; A, C. olivaceumadded; O, C. olivaceum and Bacillus sp. added.

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BIOLOGICAL CONTROL OF C. OLIVACEUM 515

Mushroom yields from the 2% malt extractexperiments (Table 1) containing olive greenmold only showed complete failure of any A.bisporus fruiting body formation. However, thecrop yields from trays containing the bacillusproduced maximum yields, even in the presenceof the chaetomium mold. Analysis of varianceindicated that treatments were significant in 2%malt extract (P < 0.05).The results for hydroponic culture with liquid

compost demonstrated significant biologicalcontrol both during the mycelial growth phase (P< 0.01; Fig. 5) and during fruiting of the mush-room, as the yields demonstrate (Table 1) (anal-ysis of variance, P < 0.05). Trays with only thecompetitor C. olivaceum added did not producemushrooms. However, similar to the 2% maltexperiments, the maximum yield occurred fortrays containing the Bacillus AOG and C. oliva-ceum together. These experiments clearly showthe benefits resulting from selective protection(biological control) through controlled fermenta-tion of the nutrient substrate.The successful use of hydroponics coupled

with pure cultures of microorganisms (as dem-onstrated in these experiments) are importantfactors in the microbiological development ofthis fermentation process. Compost manufac-ture and materials are evaluated to cost 20 to25% of the total mushroom production expendi-tures (18). Furthermore, on many larger inten-sive units, the disposal of spent compost fre-quently poses serious health hazards (e.g.mushroom workers' lung [12]). Thermophilicbacterial liquid feeding of a supporting mediumin mushroom cultivation offers a new approachto this fermentation, possibly leading to eventualcontinuous-culture techniques.The isolation of a thermophilic microorganism

antagonistic towards olive green mold is aunique finding. The application of this organismmay eventually form an effective biological con-trol method in the commercial cultivation ofmushrooms. Furthermore, the use of this orga-nism or its metabolites could potentially beextended for use in the preparation of differen-tial media or in the protection offoods and plantsfrom fungal invasion.

ACKNOWLEDGMENTS

We gratefully thank Trent Waldern of the Fraser ValleyMushroom Growers Association and D & W Mushrooms,Ltd., for mushroom compost samples and Rick Yada forstatistical analysis.

This work was supported by the British Columbia Depart-ment of Agriculture.

LITERATURE CITED1. Anonymous. 1981. Good growth potential seen for Canadi-

an mushroom industry. Food Can. 41:15.2. Atkins, F. C. 1972. Minor diseases and competitors, p.

128-135. In Mushroom growing today, 6th ed. Faber andFaber Ltd., London.

3. Beach, W. S. 1937. Control of mushroom diseases andweed fungi. Bull. 351. Pennsylvania Agricultural Experi-ment Station, State College, Penn.

4. Bels-Koning, H. C., J. P. G. Gerrits, and M. H. Vaan-drager. 1%2. Some fungi appearing towards the end ofcomposting. Mushroom Sci. 5:165-169.

5. Brancato, F. P., and N. S. Golding. 1953. The diameter ofthe mold colony as a reliable measure of growth. Mycolo-gia 45:848-864.

6. Brown, H. P. 1937. Mushroom bed invaders-their habitsand the means of control. Agric. Gaz. N.S.W. 48:436-439.

7. Davis, A. C. 1938. Mushroom pests and their control. Cir.457. U.S. Department of Agriculture, Washington, D.C.

8. Eddy, B. P., and L. Jacobs. 1976. Mushroom compost as asource offood for Agaricus bisporus. Mushroom J. 38:56-59, 67.

9. Gordon, R. E., W. C. Haynes, and C. H. Pang. 1973. Thegenus bacillus. Agric. Handb. 427. Agricultural ResearchService U.S. Department of Agriculture, Washington,D.C.

10. Hayes, W. A. 1974. Mushroom cultivation-prospectsand developments. Proc. Biochem. 9:21-24, 28.

11. Huhnke, W. 1970. Modern mushroom farming. Sci. J.6:62-66.

12. Kleyn, J. G., W. M. Johnson, and T. F. Wetzler. 1981.Microbial aerosols and actinomycetes in etiological con-siderations of mushroom workers' lungs. AppI. Environ.Microbiol. 41:1454-1460.

13. Knrtzmnan, R. H., Jr. 1979. Mushrooms: single cell pro-tein from cellulose, p. 305-339. In D. Perlman (ed.),Annual reports on fermentation processes, vol. 3. Aca-demic Press, Inc., New York.

14. Lambert, E. B. 1938. Principles and problems of mush-room culture. Bot. Rev. 4:397-426.

15. Lambert, E. B., and T. T. Ayers. 1953. Diseases of thecommon mushroom. U.S. Dep. Agric. Yearb. Agric.1953:478-482.

16. Nair, N. G., and P. C. Fahy. 1972. Bacteria antagonisticto Pseudomonas tolassii and their control of BrownBlotch of the Cultivated mushroom Agaricus bisporus. J.Appl. Bacteriol. 35:439-442.

17. Rettew, R. 1948. Manual of mushroom culture, 4th ed.Mushroom Supply Co., Taughaenamon, Pa.

18. Royse, J., and L. C. Schisler. 1980. Mushrooms: theirconsumption, production and culture development. Inter-discip. Sci. Rev. 5:324-332.

19. Sinden, J. W. 1971. Ecological control of pathogens andweed-molds in mushroom culture. Annu. Rev. Phyto-pathol. 9:411-432.

20. Stanek, M., and J. Rysava-Zatecka. 1970. Application ofselected strains of thermophilic microorganisms for thefermentation of sterilized synthetic mushroom compost.Mykol. Sb. 7:103-109.

21. Townsley, P. M. 1974. Pure cultural industrial fermenta-tion in the production of mushrooms. Can. Inst. Food Sci.Technol. J. 7:254-255.

22. Vedder, P. J. C. 1978. Diseases and pests, p. 317-392.Modern mushroom growing. Educabock B. V., Culem-borg, The Netherlands.

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