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, Isolate No. mm/wk 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 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. Plugs were placed Because the media with benomyl (BDP and near the edge of the plate. Plates were incubated BSMA; FIG. 1) had performed well, benomyl was in the dark at 25 C + 0.1 C, except for Serpula tested along with the amendments dichloran, lacrymans, which was incubated at 20 C (its max- PCNB and cycloheximide on a subsample of the imum temperature for growth is about 25 C). fungi. Dichloran alone was not effective against The longest colony radius was measured after 3 Trichoderma spp., but benomyl was (FIG. 2). or 4 da, at 1 wk, and at 2 wk. Where rhizomorphs However, dichloran, unlike benomyl, was effec- grew beyond the mycelial margins, these were tive against Mucor hiemalis Wehmer, and it did included in the measurement. Recognizing that not inhibit the tested hymenomycetes. The com- colony growth may not be linear with time for bination with 2 mg/L dichloran was more effec- all fungi, the longest possible growth period was tive than that with 4 mg/L. Therefore, BSMA BDP BSMA OPP PON PPP BDS Trametes versicolor Phellinuspini I Perenniporiasubacida H. annosum H. annosum I Ganoderma tsugae S. subtomentosum Serpula lacrymans Meruliustremellosus I Coniophoraputeana Lentinusedodes Armillariaostoyae A. mellea z A. gallica I A. gemina A. calvescens 0?C Trichodermakoningii I H T. harzianum * m P. dimorphospora I Penicilliumdiversum Cd) Fusariumnegundi Aspergillusniger Alternariaalternata I 0 Xylariapolymorpha z4 Ophiostomaminus Daldiniaconcentrica Rhizopus arrhizus Mucorramannianus 0 50 Mucorhiemalis Gongronellasp. Mortierellaspinosa M. isabellina I0 () 50 0 50 0 50 0 50 0 50 0 50 Percent of Growth on MEA

FIG. 1. Growth of test fungi (TABLE I) on various semi-selective media relative to growth on MEA. Bars are truncated above 100%. 300 MYCOLOGIA

40 [

30 -

A

20 v

10 10 I ] x ...... ---- xwtt Phellinus I pini _ ' i I 0 0 50 100 0 25 50 Percentof Growthon MEA PCNB (mg/l) FIG.2. Effectof benomyl and dichloranalone and FIG. 3. Effect of PCNB on growth of two hymen- togetheron growthof five fungi. Benomyl was used at omycetes (solid lines) and four non-hymenomycetes 2 mg/L. The basal medium was MEA. that are not effectively inhibited by benomyl and di- chloran (dashed lines). The basal medium was MEA with benomyl and dichloranat 2 mg/L each. O = Al- ternariaalternata, A = Mucorramannianus, 0 = Mor- was prepared with the addition of 2 mg/L dichlo- tierella isabellina, * = Gongronella sp., ? = Hetero- ran (BDS) and tested against all the fungi. Al- basidion annosum, * = Phellinuspini. though several Mucorales and deuteromycetes were still relatively uninhibited, Fusarium ne- from Norway spruce [Picea abies (L.) Karst.] gundi and Mucor hiemalis (one of the fastest- stumps cut 13 years before sampling with ad- growing fungi tested) were inhibited at no cost vanced white rot by Heterobasidion annosum, in hymenomycete growth (FIG. 1). Also, the fast- sugar maple (Acer saccharum Marsh.) stumps cut growing Trichoderma spp. were more inhibited 5 years before sampling with mostly advanced than by BSMA alone. white rot by Armillaria sp., a dead white pine Although the media containing PCNB did (Pinus strobus L.) with mixed decay containing not perform well (PON and PPP; FIG. 1), it was Hyphoderma praetermissum (Karst.) Erikss. & tried at several concentrations along with be- Strid, oak (Quercus sp.) stumps with brown rot nomyl and dichloran. It was at least as inhibitory by Daedalea quercina Fr., and a Norway spruce to the hymenomycetes as it was to the non-hy- log with brown rot by Fomitopsis pinicola menomycetes that were not inhibited by beno- (Swartz:Fr.) Karst. In another, smaller series, myl and dichloran alone (FIG. 3). Tricholomopsis platyphylla was isolated from Cycloheximide was also added (after auto- both rhizomorphs and decay in sugar maple claving) at 0, 1, 10 and 50 mg/L in combination stumps, and an unidentified basidiomycete was with BDS, but it prevented growth of most ba- isolated from mycelial cords. sidiomycetes at 10 mg/L, at which concentration With H. annosum and Armillaria sp., isola- non-hymenomycetes were virtually uninhibited tion efficiency was similar for all media, but BDS (data not shown). performed marginally better than the others (TA- The media that showed promise in laboratory BLE III). H. praetermissum was not isolated with experiments were compared in isolations from PPP or OPP. With the brown rots, PPP was dis- field samples. In one series, isolations were made tinctly less effective than other media. OPP WORRALL:HYMENOMYCETE SELECTION MEDIA 301

TABLEIII

EFFICIENCY OF ISOLATION OF SEVEN HYMENOMYCETES ON FIVE SEMI-SELECTIVE MEDIA Media Percentof total chips Fungi PPP BSMA BDP BDS OPP

Armillaria sp.a (6; 60)b 22 18 15 23 22 Heterobasidionannosuma (9; 60) 40 48 42 50 43 Hyphodermapraetermissum (1; 20) 0 35 22 35 0 Daedalea quercina(1; 8) 25 75 88 100 100 Fomitopsispinicola (1; 32) 50 84 75 91 66 Tricholomopsisplatyphylla (3; 12) NTc 100 100 100 0 Unidentifiedmycelial cords (2; 8) NT 75 63 88 0 a Samples of Armillariasp. and H. annosum were from stumps with very advanced,old decay, materialthat generallypresents a challengein isolation. b The numberof distinct samples is followed by the total numberof chips (or cord segments)per medium. c NT = not tested. worked well for D. quercina but less so for F. Maloy (5) showed, it is best used at concentra- pinicola. In the second series of isolations, nei- tions below 5 ppm. The present results show that ther T. platyphylla nor the unidentified basidio- a good number ofnon-hymenomycetes grow well mycete was isolated with OPP; BSMA, BDP and on BSMA, but that is because these species were BDS worked fairly well. chosen to represent the most troublesome fungi. Besides isolation efficiency, other factors are Although Heterobasidiomycetes were not ex- important in selecting a medium. Therefore, the plicitly tested, I have easily isolated a jelly fun- following observations are offered. gus, Dacrymyces stillatus Nees: Fr., on BSMA. The most fast-growing contaminants oc- Although laboratory tests suggested that beno- curred on PPP. Thus, frequent inspections were myl plus dichloran provided only an incremental necessary to rescue a desired colony from a con- improvement in selectivity over benomyl alone, taminant growing from another chip in the same the field isolations showed a clear difference with plate. This also occurred, but was generally less some materials. BDS was the best among the of a problem, with BSMA, BDP and BDS. It media tested. rarely occurred on OPP. The types of contami- For isolation of specific hymenomycetes, oth- nants differed among the media. On PPP, Tricho- er media may also serve well. For example, al- derma sp. was a common contaminant. On the though some non-hymenomycetes grew some- benomyl media, yeasts, bacteria, other basidio- what slower on BDS than on BDP, other problem mycetes and occasionally members of the Mu- fungi not tested may be better inhibited by BDP. corales were the most frequent contaminants. If the target hymenomycete is one of those that Except for BDP, only other basidiomycetes and grow well on it, BDP would then be a more suit- Mucorales threatened neighboring chips on the able medium. A richer medium than the others, same plate. On BDP, yeast growth was very heavy BDP leads to more luxuriant characteristic growth and sometimes spread across plates. of hymenomycetes that can aid in rapid identi- OPP resulted in slower growth of target fungi fication. A negative side of the richness is heavy than the other media. It was sometimes neces- growth of yeasts. sary to subculture on MEA to verify identity of The use of OPP is a special case. Although isolates on OPP. BDP generally had the most o-phenylphenol was originally suggested for gen- luxuriant growth of hymenomycetes. However, eral isolation ofbasidiomycetes (9), the medium in some cases, they were at first inhibited, pro- tested here and a similar one tested by Whitney ducing aerial mycelium on chips but not growing et al. (12) are clearly inappropriate for all but a on the agar. few. The pH seems to be within the range for of most so the inhi- DISCUSSION growth wood-decay fungi, bition is apparently due to Na-OPP itself. OPP For general isolation of hymenomycetes, be- inhibits many non-hymenomycetes as well as or nomyl is the closest to an ideal ingredient. As better than does BDS, but several, notably the 302 MYCOLOGIA

fast-growing Trichoderma harzianum, grow fast- ACKNOWLEDGMENTS er on OPP. some Armillaria Although species This project was supported by the USDA via a grew well on OPP, others were inhibited, in one McIntire-Stennis grant. L. M. Dann provided excellent case severely. OPP does have an advantage in technical assistance. Some of the isolates were kindly that isolation plates can be untended for weeks provided by R. A. Blanchette, T. C. Harrington and C. K. T. C. reviewed the manu- with less chance of than with J. Wang. Harrington being overgrown script and provided helpful advice. C. J. K. Wang as- other media. On the other hand, most hymeno- sisted in the identification of some isolates. mycetes also grow more slowly on OPP than on other media. Whitney et al. (12) isolated fewer LITERATURE CITED hymenomycetes other than Armillaria sp. on an 1. T. M. o-phenylphenol medium than on MEA. Harrington, C., M. Furniss, and C. G. Shaw. 1981. Dissemination of It should also be noted that there are hymenomycetes by many Dendroctonuspseudotsugae (Coleoptera: Scolyt- forms of media based on o-phenylphenol in the idae). Phytopathology 71: 551-554. literature. Some workers use the sodium salt (7); 2. Hunt, R. S., and F. W. Cobb, Jr. 1971. Selective others apparently use the acid form (9, 12), which medium for the isolation of wood-rotting ba- Canad. J. Bot. 49: 2064-2065. is relatively insoluble. Some media are acidified sidiomycetes. 3. Kuhlman, E. G. 1966. Recovery of Fomes an- with (7), combined with peptone (9) or used re- nosus spores from soil. Phytopathology 56: 885. duced levels of malt extract (13). 4. , and F. F. Hendrix, Jr. 1962. A selective Although PCNB is reportedly effective against medium for the isolation of Fomes annosus. 1310-1312. the Mucorales, the media containing PCNB did Phytopathology 52: 5. C. 1974. for the not well. The Maloy, O. Benomyl-malt agar perform dose-response experiment purification of cultures of wood decay fungi. Pl. showed that it was not useful in combination Dis. Reporter. 58: 902-904. with benomyl and dichloran. Harrington et al. 6. Raabe, R. D., and J. H. Hurlimann. 1971. A (1) found that 150 ppm PCNB, when added to selective medium for isolation of Armillaria mellea. Plant Pathol. No. Univ. an acidified benomyl medium, made little dif- Calif. 3-May. Calif. Agric. Ext. Serv. ference in the isolation of hymenomycetes from 7. Rishbeth, J. 1972. The production of rhizo- Douglas-fir beetles. morphs by Armillaria mellea from stumps. Eur. Not addressed explicitly in this study are J. For. Pathol. 2: 193-205. 8. D. and T. C. 1988. Root comparisons among anti-bacterial agents. Gen- Rizzo, M., Harrington. and butt rot on balsam fir and red bacteria have not been a serious fungi spruce erally, problem in the White Mountains, New Hampshire. Pl. on either BSMA or BDS. Although streptomycin Dis. 72: 329-331. is commonly used with benomyl, other antibi- 9. Russell, P. 1956. A selective medium for the otics may work better with particular field ma- isolation of basidiomycetes. Nature 177: 1038- terials. Acidification has also been used to inhibit 1039. 10. Taylor, J. B. 1971. A selective medium for the growth of bacteria, but it is not recommended isolation of basidiomycetes from diseased roots, for benomyl-dichloran media without carefully mycorrhizas, and soil. Trans. Brit. Mycol. Soc. investigating the effect on antifungal activity. In 56: 313-314. a medium made with 11. Uscuplic, M., and R. G. Pawsey. 1970. A selec- preliminary investigations, tive medium for the isolation of 2 and 200 PCNB and acid- Polyporus ppm benomyl ppm schweinitzii. Trans. Brit. Mycol. Soc. 55: 161- ified to about pH 3 was less effective against 163. Trichoderma spp. than was BSMA (data not 12. Vaartaja, 0. 1968. Wood inhabiting fungi in a shown). pine plantation in Australia. Mycopathol. My- col. 34: 81-89. In conclusion, BDS is recommended for gen- Appl. 13. R. D., D. T. Myren, and W. E. Britnell. A Whitney, eral isolation of hymenomycetes. convenient 1978. Comparison of malt agar with malt agar stock solution is prepared by dissolving 40 mg plus orthophenylphenol for isolating Armillaria Benlate 50% W.P. in 50 ml of warm 95% ethanol, mellea and other fungi from conifer roots. Ca- to 100 ml with water, and 20 nad. J. For. Res. 8: 348-351. diluting adding mg 14. and R. 1969. Mucorales- dichloran. The stock solution can be Zycha, H., Siepmann. refrigerated eine Beschreibung aller Gattungen und Arten for at least several months without loss of activ- dieser Pilzgruppe. J. Cramer, Lehre. 355 p. ity. Ten ml of the stock solution is added to one L MEA before autoclaving, after which 100 mg Accepted for publication January 14, 1991 streptomycin is added.