JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1993, p. 572-577 Vol. 31, No. 30095-1137/93/030572-06$02.00/0Copyright © 1993, American Society for Microbiology

The Benomyl Test as a Fundamental Diagnostic Methodfor Medical Mycology

RICHARD C. SUMMERBELL

Ontario Ministry of Health, Box 9000, Terminal A, Toronto, Ontario MSW IR5, and Department ofMicrobiology, University of Toronto, Toronto, Ontario, Canada

Received 22 September 1992/Accepted 22 December 1992

The fungicide benomyl has long been known to differentially affect major taxonomic groups of fungi. In thepresent study 163 species or aggregates of closely similar species of medically important fungi andactinomycetes, as well as species commonly isolated as clinical contaminants, were tested to determine theirreactions to three concentrations of benomyl. Fungi of basidiomycetous, endomycetous, and microascaceousaffinities were highly resistant, including all common yeasts and Geotrichum, Pseudallescheria, Scedosporium,and Scopulariopsis species. Also resistant were fungi of pleosporalean affinities with poroconidial anamorphs,such as Alternaria, Bipolaris, Curvularia, and Exserohilum species. Most other fungi of ascomycetous affinitywere moderately to strongly susceptible. Such fungi included dermatophytes; Coccidioides, Blastomyces, andHistoplasma species; Sporothrix schenckii; medically important aspergilli; and "black yeasts." Benomyl testingaided in the provisional identification of nonsporulating mycelia, including common basidiomycetous isolatesobtained as contaminants as well as nonsporulating Aspergillus fumigatus from pulmonary sources.

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecar-bamate] has been used as a fungicide for over 20 years. Itbecame known as a taxonomically selective fungicide whenBollen and Fuchs (3) and Edgington et al. (6) demonstratedthat it inhibited most ascomycetous fungi (including biolog-ically related anamorphic fungi also classifiable in the an-amorph division Fungi Imperfecti) but caused slight or noinhibition for most basidiomycetous and zygomycetousfungi. Since then it has been used in numerous selectivemedia for the division Basidiomycota (8), the division Zygo-mycota (17), and members of the resistant genus Alternaria(1). It has also been used to aid in the classification of fungiof uncertain taxonomic disposition, particularly nonsporu-lating entities such as the E-strain somatic anamorphs of thediscomycetous Tricharina species (4). Recently it has beensuggested that benomyl-amended medium could be used inmedical mycology as a rapid means of distinguishing thebasidiomycetous anamorph Sporothrix cyanescens from theascomycetous anamorph Sporothrix schenckii (15).

Studies on the selectivity of benomyl have shown adiverse array of resistant taxa within the division Ascomy-cota and related anamorphs. Bollen and Fuchs (3) andEdgington et al. (6) showed that a group of fungi related tothe families Pleosporaceae and Pyrenophoraceae of the classLoculoascomycetes (2), including the genera Pleospora,Pyrenophora, and Cochliobolus, mostly with poroconidialanamorphs such as Alternaia and Drechslera species, wereresistant. Also, the same authors showed that some mem-bers of the class Hyphomycetes with annellidic conidiogen-esis and some yeasts were resistant. Summerbell (16)showed that some sterile mycelia of ascomycetous affinity,such as the Mycelium radicis atrovirens somatic statesrelated to Phialocephala fortinii, could be selected on thebasis of resistance to 2 ,ug of benomyl per ml, even thoughthey were susceptible to higher concentrations of the fungi-cide.

In the present study, medically important fungi and fungicommonly isolated as contaminants in the medical labora-tory were studied to determine the extent to which an assayof benomyl tolerance could facilitate accurate identification.

In the process, patterns of taxonomic affinity among theascomycetous fungi resistant to benomyl were clarified, andit was shown that members of the class Endomycetes and ofthe family Microascaceae, both containing significant patho-genic fungi, were systematically resistant to benomyl at theconcentrations used. Mucoralean members of the divisionZygomycota, already well known to be resistant to benomyl(3, 6, 16), were not included in the present study.

MATERIALS AND METHODS

Fungi were from the culture collection of the OntarioMinistry of Health and from isolates obtained from clinicalsamples or received for identification during the course ofthe study. All but a very small number of isolates wereoriginally from human medical specimens. The balance werefrom veterinary specimens or were environmental isolates ofmedically important species.Benomyl (Benlate, wettable powder with 50% active in-

gredient; Du Pont Co., Wilmington, Del.) was incorporatedinto the general fungal growth and sporulation medium (11),modified Leonian's agar (distilled water, 1,000 ml; maltextract [Difco Laboratories, Detroit, Mich.], 6.25 g; mal-tose, 6.25 g; monopotassium phosphate, 1.25 g; yeast extract[Difco], 1.0 g; magnesium sulfate. 7H20, 0.625 g; BactoPeptone [Difco], 0.625 g; Bacto Agar [Difco], 20 g), at 0, 2,4, and 10 ,ug of active ingredient per ml. The benomyl wasmade up as a 2% suspension in sterile distilled water andaseptically added to preautoclaved medium cooled to ap-proximately 50'C but not yet solidified (9). Subcultures weremade from culture collection isolates variously maintained(under sterile mineral oil on modified Leonian's agar, frozenat -20'C on modified Leonian's or Sabouraud peptoneglucose agar, or in sterile distilled water) or from newlyobtained clinical isolates. These subcultures were grown onmodified Leonian's agar for 7 days at 25°C prior to testing.They were then inoculated to grow as single colonies at thecenter of an 85-mm-diameter petri dish. Colonies wereallowed to grow for 7 days at 25°C, and radial growthdiameters were measured. A benomyl-resistant basidiomy-

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TABLE 1. Clinical yeast isolates on benomyl

Benomyl concn % of controlSpecies (p,g/ml) causing growth on

(no. of isolates) >50% growth 10-,ug/mlinhibition benomyl

Candida albicans (3) >10 >95Candida ciferrii (1) > 10 >95Candida guilliermondii (2) > 10 >95Candida lusitaneae (4) >10 >95Candida parapsilosis (2) >10 >95Candida tropicalis (4) > 10 >95Cryptococcus hungaricus (1) >10 >95Cryptococcus neofonnans (5) >10 >95Cryptococcus uniguttulatus (1) >10 >95Hansenula anomala (2) >10 >95Kioeckera apiculata (1) > 10 >95Malassezia furfue (1) > 10 >95Malassezia pachydermatis (1) > 10 >95Pichia farinosa (1) >10 >95Rhodotorula rubra (2) >10 >95Saccharomyces cerevisiae (1) >10 >95Torulopsis candida (1) >10 >95Torulopsis glabrata (3) >10 >95Tichosporon beigelii (3) >10 >95

a Grown on medium overlaid with olive oil.

cetous isolate and a susceptible isolate of Exophiala spin-ifera were used as controls. The benomyl-free modifiedLeonian's agar plate in each trial served as a within-testcontrol of inoculum viability.

RESULTS

Because of the large number of taxa tested, the results arepreliminarily broken down according to traditional taxo-nomic groupings in Tables 1 to 5. In these tables, the data areshown in the form which proved most useful for the succinctdescription of differences between isolates: first, the lowestconcentration of benomyl causing 50% or greater inhibitionof radial growth, and second, the percentage of controlgrowth evinced on medium containing 10 ,ug of benomyl perml. Fungi which showed essentially the same growth diam-eter on 10 ,ug of benomyl per ml as on the control, withdeviations from control growth falling within the overallvariance of colony diameters produced by the species understandard conditions, are listed as growing at >95% of thecontrol diameter. Species in which some isolates weresignificantly more tolerant than others are broken down intotolerance classes. A sharp biotype distinction between theseclasses should not be inferred.Fungi showing complete resistance to benomyl at the

concentrations used included all those traditionally consid-ered yeasts (i.e., excluding "black yeasts" such asAureoba-sidium and Exophiala species) and all filamentous basidio-mycetous fungi. The basidiomycetous fungi tested were allfrom medical specimens. Such isolates, often producingnonsporulating or arthroconidial colonies, are ubiquitouscontaminants in medical specimens, particularly respiratorysecretions (14). The yeasts tested included both species ofendomycetous affinity (the Candida and Torulopsis speciestested, as well as Saccharomyces cerevisiae, Pichia fari-nosa, Kloeckera apiculata, and Hansenula anomala) andspecies of heterobasidiomycetous affinity (Cryptococcus,Trichosporon, Malassezia, and Rhodotorula species).Among the ascomycetous fungi tested, as well as among

related coelomycetous and hyphomycetous anamorphic

TABLE 2. Medically important members of the familyDematiaceae and other common dematiaceous fungi

of the clinical laboratoryBenomyl concn % of control

Species (pg/ml) causing growth on(no. of isolates) >50% growth 10-pLg/ml

inhibition benomyla

Alternaia alternata (3) >10 >95Aureobasidium pullulans (5) 2 -

Bipolaris spp. (2) >10 >95Botryosporium pulchrum (1) 10 47Cladosporium carionii (2) 2 -

Cladosporium cladosporioides (3) 2 -

Cladosporium herbarum (2) 2 -

Cladosporium sphaerospermum (1) 2 -

Curvularia sp. (1) >10 >95Dactylaria constricta (1) 4 -

Epicoccum purpurescens (1) >10 >95Epicoccum purpurescens (1) 4 -

Exophiala jeanselmei (7) 2Exophiala jeanselmei (2) 4Exophiala cf. "mansonii" (4) 10 trExophiala moniliae (2) 2 -

Exophiala pisciphila (1) 2 -

Exophiala spinifera (4) 2 -

Exophiala spp. (7) 2 -

Exserohilum rostratum (1) >10 >95Fonsecaea pedrosoi (1) 2 -

Lecythophora hoffmanii (1) 2 -

Lecythophora sp. (1) 2 -

Madurella mycetomatis (1) 2 -

Nigrospora sp. (1) 2 -

Nodulisporium sp. (1) 2 -

Oidiodendron cereale (2) 2 -

Phaeoannellomyces werneckii (2) 2 -

Phaeococcomyces sp. (1) 2 -

Phialophora cinerescens (2) 2 -

Phialophora melinii (1) 2 -

Phialophora olivacea (1) 2 -

Phialophora parasitica (3) 2 -

Phialophora repens (4) 2 -

Phialophora nchardsiae (2) 2 -

Phialophora verrucosa (3) 2 -

Phialophora sp. (2) 2 -

Pithomyces chartarum (2) 2 -

Rhinocladiella atrovirens (1) 2 -

Rhinocladiella sp. (1) 2 -

Scedosporium prolificans (2) >10 >95Scytalidium dimidiatum (11) 10 or >10 29.4-80 (avg, 56.7)Scytalidium hyalinum (4) 10 or >10 10-100Sporothrix schenckii (9) 2 -

SporothriX sp. (ophiostomatalean 2 -

anamorph)(1)Torula graminis (1) 2 -

Wangiella dennatitidis (11) 2 -

Xylohypha bantiana (1) 2 -

Xylohypha emmonsii (1) 2 -

a, no growth; tr, trace growth on inoculum plug.

fungi, several significant patterns of resistance emerged.Pleosporaceous anamorphs in the genus Alternaria andpyrenophoraceous anamorphs in the genera Bipolanis, Cur-vularia, and Exserohilum (family classification as per Barr[2]) showed their already well-attested (3, 6) resistance tobenomyl. Geotrichum candidum, a species of endomycetousaffinity, was as tolerant of benomyl as the yeast members ofthe group. All isolates with affinities to the family Microas-caceae, including Pseudallescheria boydii, Petriella setifera,Scedosporium prolificans, and Scopulariopsis and Dorato-myces species, were completely resistant to 10 ,ug ofbenomyl per ml. Other, unrelated fungi with annellidic

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574 SUMMERBELL

TABLE 3. Medically important members of the familyMoniliaceae and other common moniliaceous fungi of

the clinical laboratory on benomyl

Benomyl concn % of c(Species (,ug/ml) causing growt

(no. of isolates) >50% growth 10-,uginhibition benor

monium bacillosporum (1) 2monium sclerotigenum (1) 2monium strictum (1) 2 ]odontium salmoneum (1) 2rinium spp. (2) 2

rographis kalrae (3) 2

!rgillus flavus (3) 4 O-]!rgillus fumigatus (19) 2!rgillus fumigatus (11) 4!rgillusfumigatus (2) 4 23.8,!rgillus fumigatus (1) 10 1:wrgillus fumigatus 2

onsporulating) (2)!rgillus fumigatus 4

onsporulating) (3)rgillus glaucus ser. (2) 2'rgillus nidulans (2) 4rgillus sydowii (2) 2

'rgillus terreus (2) 2'rgillus ustus (2) 2, 4iveria bassiana (1) 2

tomyces dermatitidis (2) 2!ara sp. (2) 2,sosporium keratinophilum (1) 2isosporium cf. 2teenslandicum (1),idioides immitis (2) 2rtomyces sp. (1) >10 >9''odontium album (1) >10 9vodontium album (1) 4'ermophyton floccosum (2) 2rium aquaeductuum (1) 10 5(rium coeruleum (1) 2 2'rium equiseti (1) 10 4rium graminearum (1) 4rium oxysporum (3) 2-4rium solani (1) 4 N'rium sporotrichoides (1) 10 2'rium verticillioides (1) 2richum candidum (2) >10 >9'gplasma capsulatum var. 2psulatum (3)fodendron sp. (1) 25ranchea sp. (3) 2odochium sp. (1) 2-osporum canis (3) 2osporum gypseum (2) 2-hocola canadensis (1) 2-

ilomyces farinosus (1) 2

ilomyces inflatus (1) 2ilomyces lilacinus (1) 4 N'ilomyces variotii (1) 4cillium aurantiogriseum (1) >10 >gccillium canescens (1) 2cillium chrysogenum (1) 2cillium citreonigrum (1) 2 N7cillium griseofulvum (1) 2oulariopsis brevicaulis (3) >10 >95,ulariopsis sp. (2) >10 >95donium chrysospermum (1) 2'ophyton ajelloi (1) 2cophyton equinum (1) 2cophyton megninii (1) 2

-ontrolth ong/gmlrMyla

1.0

*10

33.31.8

TABLE 3-Continued

Benomyl concn % of controlSpecies (p.g/ml) causing growth on

(no. of isolates) >50% growth 10-pg/mlinhibition benomyla

Trichophyton mentagrophytes (7) 2Trichophyton tonsurans (1) 2Trichophyton rubrum (5) 2Trichophyton schoenleinii (1) 2Trichophyton terrestre (1) 2Trichophyton verrucosum (1) 2Trichophyton violaceum (1) 2Verticillium dahliae (1) 2Verticillium sp. (1) 2 tr

a no growth; tr, trace growth on inoculum plug; NT, not tested.

- conidiogenesis, such as Exophiala species, were susceptibleto benomyl at this level.

- Fungi related biologically to the order Onygenales were

universally highly susceptible to benomyl. This group in-- cluded dermatophytes; Histoplasma, Coccidioides, and

Blastomyces species; Myxotrichum deflexum; Malbranchea1.0 species; and the tested species of the genus Chrysosporium.tr Certain species which were partially or completely inhib-

ited by 10 ,ug of benomyl per ml showed a degree of- resistance to lower concentrations of the fungicide. The- skin- and nail-infecting Scytalidium hyalinum and Scytalid-- ium dimidiatum (synanamorph of Nattrassia mangiferae,- formerly Hendersonula toruloidea) showed a moderate de-

gree of resistance. The genera Aspergillus and Fusarium5 tended to have some partially resistant isolates, as well as

;6.5 many highly susceptible isolates. Radial growth of all iso-lates was inhibited by 50% or more by 10 ,ug of benomyl perml. Members of the genus Penicillium, normally highly

L0.0 susceptible to benomyl (3, 6, 16), were mainly susceptible in7.8 the present study. However, one isolate of Penicillium3.5 aurantiogriseum was completely resistant to the concentra-- tions used.T5.0

15

T

Continued

TABLE 4. Medically important and other clinically isolatedmembers of the Ascomycota and Coelomycetes on benomyl

Benomyl concn % of controlSpecies (,ug/ml) causing growth on

(no. of isolates) >50% growth 10-,ug/mlinhibition benomyla

AscomycotaAphanoascus fulvescens (1) 2Ascotricha sp. (1) 2Chaetomium bostrychodes (1) 2Chaetomium globosum (2) 2Myxotrichum deflexum (2) 2Neosartorya fischeri (2) 2 -,14Ophiostoma stenoceras (1) 2-4Petriella setifera (1) >10 >95Pseudallescheria boydii (5) >10 >95

CoelomycetesConiothyrium sp. (1) >10 >95Lasiodiplodia theobromae (1) 2 11.8Pestalotia sp. (1) 2Phlyctaena sp. (1) 2Phoma cf. pomorum (1) 2Pyrenochaeta unguis-hominis (1) 10 10

,no growth.

AcreAcre)Acre)AcroArth/ArthiAspeAspeAspeAspeAspeAspe

(n(Aspe

(n(AspeAspeAspeAspeAspeBeauBlastChalChryChryqu

CoccDoraEngyEngyEpid,FusaFusaFusaFusaFusaFusaFusaFusaGeotHist

cajHyalMalbMicrMicnMicrOnycPaecPaecPaecPaecPeni4PenicPenicPenixPenicScop,ScopISepe,TrichTrichTrich

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USE OF THE BENOMYL TEST IN MEDICAL MYCOLOGY 575

TABLE 5. Other medical mycology laboratory organisms onbenomyl, including some nonfungal organisms falling within the

technical traditions of many medical mycology laboratories

Benomyl concn % of controlSpecies (4g/ml) causing growth on

(no. of isolates) >50% growth 10-pLg/mlinhibition benomyla

BasidiomycotaRhizoctonia sp. (1) >10 >95Sclerotium sp. (1) >10 >95Sporothrci cyanescens (3) >10 >95Sporotrichum pruinosum (1) >10 >95Sporotichum sp. (1) >10 >95Unidentifiable basidiomycet- >10 >95

ous isolates (28)Ustilago sp. (1) 2

ActinomycetesActinomadura madurae (1) >10 >95Nocardia asteroides (2) >10 >95Nocardia brasiliensis (1) > 10 >95Nocardia farcinica (1) >10 >95Nocardia nova (1) >10 >95Nocardia otitidiscaviarum (1) >10 >95Rhodococcus bronchialis (1) >10 >95Rhodococcus equi (1) >10 >95Rhodococcus ruber (1) >10 >95Streptomyces griseus (1) >10 >95Streptomyces lavendulae (1) >10 >95Streptomyces nmosus (1) >10 >95

Algae: Prototheca stagnora (1) >10 >95

a _, no growth.

DISCUSSION

In practical diagnostic medical mycology, workers have

seldom had access to convenient characters which would

reflect the fundamental phylogenetic affinities of the organ-isms with which they dealt. Such characters, if accessible,are convenient clues to the identity of nonsporulating and

other difficult-to-identify fungi and may also, depending on

the homogeneity of the group involved, allow the predictionof factors beyond the scope of this paper, such as antimy-cotic susceptibility and serologic cross-reactivity. The diaz-

onium blue B staining test for yeasts (7) was one available

test yielding results predictive of phylogenetic affinity, but in

diagnostic work its use was largely preempted by the less

phylogenetically revealing but easier-to-handle Christensen

urease test. The general cycloheximide tolerance of onyge-nalean fungi, including many traditional pathogens, was

clear, but the remaining cycloheximide-tolerant fungi com-

monly seen in medical mycology did not form clear patternsof phylogenetic affinity. Septal pore ultrastructure, a majorcharacter used in formal mycologic taxonomy to investigatethe classification of ascomycetous and basidiomycetousfungi between the levels of phylum and family, was imprac-tical for diagnostic laboratory use. Benomyl tolerance at first

glance seemed an unlikely candidate for routine use in

identifying the fundamental affinities of unknown isolates,

mainly because of the diverse minority of ascomycetousfungi which were known to be resistant. The present study,by showing that the highly resistant fungi mainly fall into a

few well-circumscribed classes and families, attempts to

render benomyl testing useful for a wide variety of medically

important fungi and species commonly encountered as clin-

ical contaminants.

The rapid identification of filamentous basidiomycetousfungi in clinical specimens is one major advantage of thistechnique. These fungi are among the most common sterilemycelia isolated from clinical materials and also among themost common normally nonpathogenic fungi producing ei-ther rhexolytically or schizolytically seceding arthroconidia(14). With but a few exceptions, they lack clamp connections(14). In recent decades they have been easily distinguishedfrom anamorphs of onygenalean affinity, such as Coccid-ioides, Histoplasma, and Blastomyces species, by theirpronounced intolerance of 100 ,ug of cycloheximide per ml.More difficult, however, has been the task of distinguishingthem from cycloheximide-susceptible isolates. The mostclinically important of these are the "dysgonic," or initiallynonsporulating, isolates ofAspergillus fumigatus frequentlyisolated from patients with aspergilloma and other chroniccolonizations of the respiratory tract. As Table 3 shows,these isolates are strongly intolerant of benomyl and are thusreadily separable from nonsporulating basidiomycetousfungi. The good growth of the A. fumigatus isolates at 45°Cin vitro is also a distinguishing factor, but a few fungi ofbasidiomycetous affinity, such as Sporotrichum pruinosum,are also able to grow at that temperature.

Sterile basidiomycetous fungi may also be difficult to

distinguish accurately from sterile ascomycetous isolatesand slow-to-sporulate isolates of such fungi as Arthnnium,Nigrospora, and Phlyctaena species. The benomyl testmakes the basidiomycetous fungi easily distinguishable fromthese fungi and allows a laboratory to provide a more

informative identification than the traditional "sterile myce-lium." The benomyl test may not, however, distinguishbasidiomycetous fungi from slow-to-sporulate, benomyl-tol-erant isolates of Epicoccum purpurescens (Tables 2 and 5).E. purpurescens colonies, when nonsporulating, may betentatively distinguished by their characteristic golden andred pigments.

Arthroconidial basidiomycetous anamorphs may some-

what resemble morphologically two medically importantspecies, Scytalidium hyalinum and Arthrographis kalrae.The former fungus tends to be somewhat resistant to

benomyl; one isolate in the present study, although slightlyretarded by benomyl, managed to grow to the edges of an

85-mm-diameter petri plate of 10-Rg/ml benomyl mediumwithin 7 days. Benomyl susceptibility is thus a potentialexcluding character for this species vis-a-vis basidiomycet-ous fungi: that is, any isolate which is strongly inhibited maybe ruled out as basidiomycetous, but all benomyl tolerantisolates queried as Scytalidium hyalinum must be distin-guished by other means. The situation with Arthrographiskalrae is less complex: this species is highly susceptible to

benomyl. The benomyl test does not, unfortunately, assistwith the distinction of arthroconidial basidiomycetous iso-lates and Geotrichum candidum. In this case, however,correct morphological identification of the flattened, slimycolonies of Geotrichum candidum is not difficult (14).Some filamentous basidiomycetous fungi may produce

blastic anamorphs, such as those found in Sporothrix cy-anescens, or aleurioconidia, such as those found in Sporo-trichum spp. Sporothrix cyanescens can easily be distin-guished from Sporothrix schenckii and other Sporothrixspecies of ophiostomatalean affinity by this test (15); simi-

larly, the benomyl-tolerant species of the genus Sporotri-chum can easily be distinguished from Chrysosporium spe-cies.

Pleosporaceous or pyrenophoraceous anamorphs such as

Altemaria and Bipolaris species are occasionally seen in the

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576 SUMMERBELL

diagnostic laboratory as persistently nonsporulating isolates,even when sporulation media are used. With the benomyltest, these fungi are easily distinguished from benomyl-susceptible, fast-growing, nonsporulating dark colonies suchas young or degenerate colonies of Lasiodiplodia theobro-mae or ubiquitous plant-associated sterile mycelia such as

the so-called Mycelium radicis atrovirens (16). A minority ofPhoma and Coniothyrium species have affinities with mem-

bers of the genus Pleospora (12) (but note that Barr [2] hassuggested that Pleospora species with coelomycetous an-

amorphs might be more closely related to the genus Cur-reya), and the one such species tested, Phoma betae, hasbeen shown to be resistant to benomyl (3). The greatmajority of common Phoma and Coniothyrium (sensu lato)species, such as those with teleomorphs in the genus Lepto-sphaeria, are highly susceptible to benomyl (3). Benomyl-resistant members of the class Coelomycetes seen in thelaboratory, such as the Coniothyrium sp. in Table 4, may bebiologically related to the more common poroconidial an-

amorphs of the family Pleosporaceae or to a benomyl-resistant group not yet rigorously delimited.The benomyl tolerance of the family Microascaceae and

related anamorphs suggests that benomyl-supplemented me-dium could be used by laboratories wishing to facilitateidentification of Scedosporium or Scopulaniopsis species.Likewise, ecological or epidemiological studies in whichfungi such as Pseudallescheria boydii are being specificallyinvestigated would likely benefit by incorporating 10 ,ug ofbenomyl per ml in isolation media.The class Endomycetes, a group of organisms distin-

guished in electron microscopy by their multiperforatesepta, appears thus far to be generally resistant to benomyl;the species tested here are related to a variety of theconstituent families of the class. The ability to separateendomycetous fungi from the mainstream of ascomycetouscultures (including biologically related Fungi Imperfecti) isuseful in some situations. Candida ciferrii, with its some-

times-predominant Sporothrix catenata synanamorph, may

be distinguished from Sporothrix species related to the orderOphiostomatales by its benomyl tolerance. The benomyl-susceptible Arthrographis kalrae often grows as a yeast on

initial isolation before forming its arthroconidial mycelialphase and may easily be distinguished by benomyl testingboth from Candida species and other endomycetous yeasts

and, later, from the arthroconidial Geotrichum candidum.My reference laboratory has occasionally received fungisuch as Sporothrix schenckii, Fusarium dimerum, Lecytho-phora spp., and Aureobasidium pullulans tentatively identi-fied as yeasts, often with the remark "API 20C-no code."These slimy-spored, glabrous, and in some cases dimorphicmembers of the class Hyphomycetes may be distinguishedfrom endomycetous and heterobasidiomycetous "true"

yeasts by their benomyl susceptibility as well as by theirmorphology.Two cautions must be given about the use of benomyl

media in diagnostic situations. The first is that some plant

pathogens repeatedly exposed to benomyl have tended to

develop resistant populations (5) and some saprobic fungi in

heavily exposed areas may have been similarly affected.

Artificial mutagenesis readily yields benomyl-resistant mu-

tants in such normally highly susceptible genera as Tri-choderma (13). The appearance in the present study of a

Penicillium aurantiogriseum isolate resistant to 10 ,ug of

benomyl per ml may reflect acquired resistance due to

environmental exposure. The appearance of a tolerant Epi-

coccum purpurescens isolate may also betoken the same

phenomenon. It was because of these findings that we testeda large number of A. fumigatus isolates, especially dysgonicisolates from respiratory colonizations, in order to ascertainthat resistance to 10p,g of benomyl per ml was not present toa significant degree in this medically critical species. Al-though no such resistance was found in this study, benomyltesting clearly should be used with due caution, especiallywhen the organism queried is potentially consistent withcommon plant pathogens or food spoilage organisms such as

Fusanum and Verticillium spp.The second caution is that higher concentrations of

benomyl may significantly inhibit the organisms listed hereas tolerant. Numerous basidiomycetes show a growth inhi-bition of 50% by concentrations of benomyl below 31 ,ug/ml(6); for example, Copninus cinereus is inhibited by 40% at 15,ug/ml and by 93% at 20 ,ug/ml (10). Hence, benomyl mediashould be quality controlled to ensure that the fungicide hasbeen accurately added to media. It should be noted that afew basidiomycetous fungi are inhibited to 50% by benomylconcentrations between 2 and 10 ,ug/ml (6, 9). The group ofbasidiomycetous fungi often found as clinical contaminantsappears have little or no overlap with these species, the mostsusceptible of which are mycorrhizal symbionts whosespores do not germinate on ordinary fungal growth media.We have not obtained any basidiomycetous isolates suscep-tible to 10 ,ug of benomyl per ml in over 3 years of routinetesting in our diagnostic service.

ACKNOWLEDGMENTS

Thanks to Myrna deCastro and Judy Clent for painstaking tech-nical assistance, Margaret Kwok for medium preparation, and JuliusKane for faithful encouragement. Paul Lehmann is thanked for hiskind provision of isoenzyme characterizations to clarify the status ofsome Exophiala and Wangiella isolates. The University of AlbertaMicrofungus Collection and the American Type Culture Collectionare thanked for the provision of isolates.

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