Mycologia,83(3), 1991, pp. 296-302. ? 1991, by The New York BotanicalGarden, Bronx, NY 10458-5126 MEDIA FOR SELECTIVE ISOLATION OF HYMENOMYCETES JAMESJ. WORRALL College of EnvironmentalScience and Forestry, State Universityof New York,Syracuse, New York13210 ABSTRACT The growth rates of 32 hymenomycetesand non-hymenomyceteswere compared on five media that have been devised for isolation of hymenomycetes.A medium with 2 mg/L benomyl permitted virtuallyuninhibited growth of hymenomyceteswhile stronglyinhibiting many non-hymenomycetes, but membersof the Mucoralesand severalother species still grewwell. Testingof variouscombinations and concentrationsof ingredientsled to the use of 2 mg/L benomyl and 2 mg/L dichloranas antifungal ingredients.This was tested in isolations from various field materialsand in every case performedas well as or better than other commonly used media. Key Words: hymenomycetes,selective media, benomyl, orthophenylphenol Partly because of their unique ability to rap- mg/L (10) and, along with dichloran and phenol, idly degrade wood, hymenomycetes are an eco- at 8 mg/L (2). Maloy (5) showed that at 5 mg/L nomically and ecologically important group of and above, benomyl was inhibitory to some hy- fungi. They are among the most damaging patho- menomycetes, but at 1 mg/L it inhibited several gens of woody plants, cause losses of wood prod- deuteromycetes and had little or no effect on a ucts in service, and probably decompose the ma- large number of hymenomycetes. It has been ef- jority of the fixed carbon in terrestrial ecosystems. fectively used at 2 mg/L (8). There is also a growing interest in biotechnologi- The objective of this study was to determine cal application of these fungi in the wood-using which is the best isolation medium for hymeno- industries. mycetes. Combinations and concentrations of Although many media have been devised for selective ingredients were explored in an effort the semi-selective isolation of hymenomycetes to improve described media. (1-12), no comparisons have been reported that would allow one to rationally choose among them. MATERIALS AND METHODS o-Phenylphenol was recommended for gen- Cultures. -Wood-decay hymenomycetes were eral isolation ofbasidiomycetes (9), and has been chosen for testing based on their economic im- used more recently for isolating Armillaria spe- portance and taxonomic diversity (TABLEI). Non- cies (7, 13). Kuhlman and Hendrix (4) felt that hymenomycetes were chosen to represent im- it was too inhibitory to Heterobasidion annosum portant wood-inhabiting groups and in particular (Fr.) Bref. and devised a medium using peptone those that commonly interfere with isolation of as a carbon source with pentachloronitrobenzene hymenomycetes on selective media. (PCNB) as the main antifungal ingredient. Vaar- taja (12), apparently independently, combined Media. -In some original formulae for media, PCNB and o-phenylphenol along with the anti- the chemicals used or methods of sterilization fungal antibiotic nystatin in a medium that was are ambiguous. For clarity and convenience, the used for isolating a variety of basidiomycetes. formulae that I used are described together here. An acidified medium containing o-phenylphenol Media were autoclaved for 30 min and cooled along with copper-chrome-arsenate (a wood pre- to about 50 C before any post-autoclave ingre- servative) was devised for isolation of Phaeolus dients were added. Twenty-five ml of medium schweinitzii (Fr.) Pat. from soil (11). However, was dispensed in 90-mm-diam Petri plates. it was intentionally inhibitory to other hymeno- BDP (2): One liter of potato dextrose agar mycetes. (Difco) prepared according to label directions, Benomyl has been used at 15 mg/L for iso- plus (added after autoclaving) 5 ml of a stock lating Armillaria species (6). For general isola- solution containing 1 g phenol, 0.32 g Benlate tion of basidiomycetes, it has been used at 5 50% W.P. (50% benomyl), and 0.16 g dichloran 296 WORRALL:HYMENOMYCETE SELECTION MEDIA 297 TABLE I ISOLATESUSED, THEIR TAXONOMICAFFINITIES, AND THEIRGROWTH RATES ON MEA Growth on MEA, Fungus Isolate No. mm/wk Mucorales Mortierellaceae Mortierellaisabellina Oud. & Koning P62a 25 M. spinosa Linn. N11 53 Mucoraceae Gongronellasp. P123a 24 Mucorhiemalis Wehmer 158a 82 M. ramannianusbMoller N10 31 Rhizopusarrhizusb Fischer N12 175 Ascomycetes Daldinia concentrica(Bolt.:Fr.) Ces. & DeNot. N7c 70 Ophiostomaminus (Hedgc.)Syd. & P. Syd. N4d 19 Xylariapolymorpha (Pers. ex Mer.) Grev. N5 22 Deuteromycetes Alternariaalternata (Fr.) Keissler CAR27Aa 40 Aspergillusniger v. Tiegh. CAR32a 31 Fusarium negundiSherb. N6 32 Penicilliumdiversum Raper & Fennell D16a 13 Phialocephaladimorphospora Kendrick P109a 12 Trichodermaharzianum Rifai EP22a 105 T. koningii Oud. P947a 137 Agaricales Armillariacalvescens Berube & Dessureault 80 3 A. gemina Berube& Dessureault 64 5 A. gallica Marxm. & Romagn. 97 9 A. mellea (Vahl.:Fr.)Kummer 312 4.5 A. ostoyae (Romagn.)Herink 29 3.5 Lentinusedodes (Burk.) Sing. 301e 22 Aphyllophorales Corticiaceaes.l. Coniophoraputeana (Schum.:Fr.)Karst. 61 34 Meruliustremellosus Fr. 63f 41 Serpula lacrymans(Wulf.:Fr.) Schroet. 28 2.5 Stereaceae Stereumsubtomentosum Pouzar 5 24 Polyporaceaes.l. Ganodermatsugae Murr. 58 11 Heterobasidionannosum (Fr.) Bref. 31 47 H. annosum 41 33 Perenniporiasubacida (Pk.) Donk 2 32 Phellinuspini (Thore.:Fr.)A. Ames 4 11 Trametesversicolor (L.:Fr.) Pilit 1 45 a Provided by C. J. K. Wang;her numbersused. b Sensu Zycha and Siepmann(14). c Provided by K. E. Hammell. d Provided by T. C. Harrington;his no. C189. e Purchasedfrom the culturecollection of PennsylvaniaState University;their no. WC 305. fProvided by R. A. Blanchette;his no. 23. (2,6-dichloro-4-nitroaniline) per 100 ml 50% 2.5 N (18.7%) lactic acid and 4 ml 1% Na-OPP ethanol. The stock solution was refrigerated for (2-phenylphenol, sodium salt tetrahydrate). up to two weeks. PPP (3, 4): 5 g Bacto peptone, (Difco) 20 g MEA: 15 g dried malt extract (Difco) and 15 agar, 0.25 g MgS04-7H20, 0.5 g KH2PO4, 0.2 g g agar per L of water. pentachloronitrobenzene (PCNB) and 0.13 g so- BSMA (8): MEA plus 10 ml Benlate stock (40 dium deoxycholate per L of water, plus (added mg/100 ml H20) and (added after autoclaving) after autoclaving), 50 mg penicillin G, 1.3 ml 100 mg streptomycin sulfate. The Benlate did 85% lactic acid, and 20 ml 95% ethanol. not completely dissolve in the stock solution or PON (12): 2 g sucrose, 10 mg PCNB, 10 mg medium. Na-OPP, and 12 g agar per L of water, plus (add- OPP (7): 30 g malt extract and 15 g agar per ed after autoclaving) 50 mg streptomycin sulfate L of water, plus (added after autoclaving) 6.6 ml and 8 mg nystatin (mycostatin). 298 MYCOLOGIA TABLEII used to calculate radial growth in mm/wk. Thus, COMPARISONOF ANTIFUNGALINGREDIENTS AND for very fast fungi, which covered the plate in 4 CONCENTRATIONSIN THE MEDIA TESTED da, growth rate was calculated from 3 da growth; Media for the slowest fungi it was calculated from 2 wk growth. Concentration (mg/L) Ingre-~Ingre- dients BDP BSMA OPPa PON PPPa BDS Isolations from field material. -To further dis- criminate among media that performed reason- 8 2 - - - benomyl 2 ably well in tests, several series of dichloran 8 - - - - 2 laboratory isolations were made. Several phenol 50 - parallel samples Na-OPP - - 40 10 - - of white and brown rots, mycelial cords of an PCNB - - - 10 200 - unknown basidiomycete, and rhizomorphs of - - - - - nystatin 8 Tricholomopsis platyphylla (Pers.: Fr.) Sing. were a Acidified. used. The decayed wood was split to expose a clean surface before chips were removed. Cords and rhizomorphs were soaked for 1 min in an Concentrations of the antifungal ingredients aqueous solution of 10% bleach/10% ethanol be- are summarized in TABLEII. pH was measured fore isolation. Chips of wood or segments of cords on solidified media using a flat-surface pH elec- or rhizomorphs were subdivided and one portion trode. Results were: MEA, 5.7; BSMA, 5.5; BDP, put on each medium. 5.5; PPP, 4.2; OPP, 3.8; PON, 6.2. Based on results with those published media, RESULTS several new combinations and concentrations of Relative to growth on MEA, PON greatly antifungal ingredients were tested. In one exper- inhibited almost all the fungi tested (FIG. 1). PPP iment, MEA was amended with 2 mg/L benomyl moderately inhibited most fungi, with no ap- (added as in BSMA, above), 4 mg/L dichloran parent selectivity for hymenomycetes. Several (added as a stock solution of 10 mg in 50 ml of Armillaria species grew well on PPP because rhi- 50% ethanol), 2 mg/L benomyl plus 4 mg/L dich- zomorph growth was stimulated. However, fast- loran, or 2 mg/L benomyl plus 2 mg/L dichloran. growing non-hymenomycetes were only partially It is known that benomyl retains activity after inhibited on PPP. OPP strongly inhibited most autoclaving (5). A preliminary experiment fungi, but several Armillaria spp. and a few other showed that autoclaving also had no effect on species grew fairly well. OPP inhibited the Mu- the antifungal activity of dichloran (data not corales, but some deuteromycetes were only par- shown). In another experiment, MEA plus 2 mg/ tially inhibited. BSMA permitted excellent growth L benomyl plus 2 mg/L dichloran was amended of hymenomycetes and more or less completely with 0, 10 or 50 mg/L PCNB before autoclaving. inhibited some non-hymenomycetes, including Finally, BDS (see Results and Discussion) was the fast-growing and ubiquitous Trichoderma spp. amended with 0, 1, 10 or 50 mg/L cyclohexi- However, it was not effective against the Mu- mide, added as an aqueous stock solution after corales and several deuteromycetes. BDP was autoclaving. additionally effective against Fusarium negundi Growth tests. -Media were inoculated with Sherb., but strongly inhibited a few hymeno- 4-mm-diam plugs of mycelium taken from the mycetes. edge of 3- to 4-wk-old colonies.