APPLIED MICROBIOLOGY, Sept. 1973, p. 271-278Copyright 0 1973 American Society for Microbiology

Vol. 26, No. 3Printed in U.S.A.

Mycotoxin-Producing Strains of Penicilliumviridicatum: Classification into SubgroupsA. CIEGLER, D. I. FENNELL, G. A. SANSING, R. W. DETROY, AND G. A. BENNETT

Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,Peoria, Illinois 61604

Received for publication 25 May 1973

Fifty-two isolates of Penicillium viridicatum Westling were divided into threegroups based on ability to produce ochratoxin and/or citrinin, color, growth rate,type of growth, odor, and isolation source. Members of group I resemble one ofthe representative strains of P. viridicatum described in the literature; thosebelonging to group II differ from group I strains in several characteristics; group

HI is a heterogeneous series of highly variable isolates. Although three subgroup-ings can be recognized, retention of all isolates in the species P. viridicatum isdeemed most appropriate at this time. Spore macerates of all isolates were

examined for virus-like particles but none were detected.

Various isolates identified as Penicillium viri-dicatum Westling are capable of producing oneor more mycotoxins including ochratoxin (5,15), citrinin and oxalic acid (8), penicillic acid(7), a hepatorenal toxin (3), and a substancecausing photosensitization in mice (4). How-ever, other isolates appear to produce no de-monstrable mycotoxins.A limited number of reports indicate that

fungal secondary metabolite synthesis, includ-ing mycotoxins, may be affected by the pres-ence of virus-like particles (VLP) (1, 2, 11). VLPhave been observed throughout the cytoplasmin thin sections of hyphae and spores of P.stoloniferum and P. brevi-compactum (9).The purpose of our experiments was to deter-

mine if there was any correlation betweencultural characteristics, mycotoxin synthesis,and possible occurrence of VLP in 52 isolates ofP. viridicatum.

This investigation was presented at the 73rdannual meeting of the American Society forMicrobiology, Miami Beach, Florida, May 6 to11, 1973.

MATERIALS AND METHODSCultures. Cultures were obtained from the Agricul-

tural Research Service Culture Collection of theNorthern Laboratory and from the Centraalbureauvoor Schimmelcultures, Baarn, Netherlands. Duringthe course of the investigation, cultures were main-tained on Difco yeast malt (YM) agar slants.

For taxonomic studies, cultures were three-pointinoculated on Czapek solution agar (Cz) and Blakes-lee's malt agar (BMA) in 100-mm petri dishes:Cz: sucrose, 30 g; NaNO,, 2 g; K2HPO4, 1 g;

MgSO4.7H2O, 0.5 g; KCl, 0.5 g; FeSO4.7H3O, 0.01 g;agar, 15 g; water, 1 liter; BMA: malt extract, 20 g;peptone, 1 g; dextrose, 20 g; agar, 20 g; water, 1 liter.Cultures were incubated 10 to 14 days at 25 C.To obtain quantities of conidia for VLP analyses,

the fungi were inoculated from slants onto whitebread cubes (1 cm3) sterilized for 10 min at 121 C in300-ml Erlenmeyer flasks. The bread had no preserva-tives added and was purchased at a local bakery.After 10 days of incubation at 28 C, a single heavilymolded cube was added to 225 to 250 g of sterile cubedbread in 2.8-liter Fernbach flasks; flasks were againincubated for 10 days. Conidia were recovered by theaddition of 500 ml of 1: 10,000 sterile aqueous TritonX-100, gentle agitation to dislodge conidia, and filtra-tion through cheesecloth. Spore suspensions werecentrifuged and washed twice with sterile water toremove starch. Approximately 18 to 22 g (wet weight)of spore paste (5-6 g dry weight) were recovered perflask.VLP detection. Sufficient spore paste to give 1 g

dry weight was suspended in 45 ml of 0.1 M phosphatebuffer, pH 7.2. Suspensions were placed in 75-ml glassBronwill flasks containing 45 g of 0.5-mm glass beadsand homogenized for 2 min at 4,000 rpm with aBronwill mechanical cell homogenizer (Braun ModelMSK) bathed in a CO, stream; stream flow wasadjusted to prevent freezing of the flask contents.Spore suspensions and homogenizing flasks were keptcold in an ice bath prior to processing. Homogenizedspores were examined under the microscope to deter-mine extent of spore rupture and then were cen-trifuged at 8,000 rpm (7.7 x 103 g) to remove sporedebris; the clear supernatant fluid was recentrifugedat 32,000 rpm (10.5 x 104 g) for 2.5 h. The supernatantfluid was discarded and the pellet was suspended in1.5 ml of phosphate buffer. This suspension wasfiltered through a membrane filter (0.45 um pore size;Millipore Corp.); a drop was placed on a Formvar-

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coated copper grid and allowed to dry. A drop of 1%uranyl acetate was placed on the grid and blotted offafter 10 s. The grid was rinsed three times withdistilled water, air-dried, and examined under anRCA EMU-3 electron microscope for the presence ofVLP.

Mycotoxin analyses. A 500-ml amount of YESbroth (2% Difco yeast extract plus 4% sucrose) inFernbach flasks was inoculated with one cube ofmolded bread per flask and incubated at 28 C as stillcultures for 8 to 10 days. The broths were decanted,adjusted to pH 2.5 with HCl, and extracted twice withan equivalent volume of CHClI. The solvent, afterbeing dried with anhydrous Na2SO4, was removed byflash evaporation, and the residual solids were dis-solved in 2 ml of CHClI. Portions (10 Mliters) werespotted on Brinkmann Silica Gel N-HR thin-layerchromatographic- (TLC) plates, along with penicillicacid, citrinin, and ochratoxin reference samples, andthe plates were developed in chloroform-ethyl ace-tate-formic acid (60:40:1). Plates were examinedunder ultraviolet light (UV) for ochratoxin and cit-rinin which fluoresce green and bright yellow, respec-tively, and then were exposed to ammonia to producea blue fluorescent penicillic acid derivative (6). Theyellow fluorescence of citrinin tends to mask the greenfluorescence of ochratoxin since both, when present,are at about the same R,. However, citrinin ceases tofluoresce after ammonia exposure, revealing an in-tense blue-fluorescing ochratoxin-ammonia deriva-tive.

Penicillic acid was confirmed by reactions withboth ammonia and with diphenylboric acid etha-nolamine to give blue-fluorescing derivatives; ochra-toxin A by reaction with ammonia to give a blue-fluorescing derivative and by methyl ester formation(12); citrinin by reaction with FeCl, to give an orangederivative. The R, values were also compared byTLC in other solvent systems (7).GLC of volatiles. Cultures were grown in bottles

for 10 days on BMA, barley, and corn; the bottles werestoppered, and the headspace was sampled by meansof a 2-ml pressure-type syringe; 1 ml of gas wasinjected onto a gas-liquid chromatography (GLC)column. Separation of volatile substances was carriedout with a Bendix model 2500 gas chromatographequipped with a flame ionization detector. Columns(1.83 by 2 mm, inner diameter) packed with Poropaktype P, 80/100 mesh (Waters Associates), were tem-perature programmed from 50 to 120 C at 5 C/min.Nitrogen was used as the carrier gas at a flow rate of50 ml/min. Injection port and detector were 210 and250 C, respectively.

Coupled GLC-mass spectrometry (MS) (DupontModel CEC) was used to obtain mass spectra of themajor substance produced by P. viridicatum NRRL5572. This compound was identified by comparing itsGLC retention time and mass spectrum to those of anauthentic standard.

RESULTS AND DISCUSSIONAnalyses for VLP. None of the 51 strains of

P. viridicatum examined revealed the presence

of VLP. It is possible, however, that VLP couldbe found if even larger masses of conidia ormycelia were examined. However, it appearsunlikely that such low concentrations of VLPswould, should they occur, affect secondarymetabolite synthesis by P. viridicatum.Culture characteristics and mycotoxin

synthesis. All isolates used in this survey hadbeen identified as P. viridicatum by one or moreinvestigators. Identification was based on Raperand Thom's Manual of the penicillia (13) inwhich species diagnoses are based primarily oncultural and morphological characteristics dis-played on Cz. Differences on BMA are notemphasized as diagnostic. Survey isolates grow-ing on this latter medium could be arrangedinto several groups on the basis of color, texture,growth rate of colonies, and odor. This separa-tion was supported by a comparison of theoriginal source of the isolates and their ability toproduce ochratoxin and citrinin.Group I. The 22 strains in group I (Table 1)

were isolated primarily from plant sources andproduced no detectable ochratoxin or citrinin.Intraperitoneal injection of the CHCl, extractsof the culture broths (YES medium) into miceresulted in no noticeable toxicity. One culture(NRRL 5570) produced penicillic acid and ap-pears to be intermediate between groups I andII; the colony pattern and odor on BMA are asgroup I, on Cz as group II.

In morphological and cultural characters onCz and BMA (Fig. 1), these isolates duplicateNRRL 963, cited by Raper and Thom (13) as theprimary culture used in their description of P.viridicatum and shown, on Cz, in color plateVIII of the Manual. On BMA, colonies areplain, rapidly and heavily sporing, thinning,and granular or slightly fasciculate at the mar-gins, attaining a diameter of 6 to 6.5 cm in 2weeks at 25 C. Conidia are bright yellow-greenwhen young, quickly becoming forest green andremaining so in age; conidial chains are incolumns, shattering easily; reverse is yellow-gold. Odor is penetrating, woodsy, earthy,Streptomyces-like, stifling. Penicilli are largeand asymmetrically branched and indistin-guishable from many other species in the Fas-ciculata.

Isolates conforming with this description areamong the most common penicillia isolatedfrom moldy grains. They represent the yellow-green end of the P. viridicatum-martensii con-tinuum discussed under taxonomic implica-tions.Group II. The 17 cultures in this group

(Table 2) also originated from plant sources andmost produced both ochratoxin and citrinin;

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TABLE 1. Characteristics of P. viridicatum, group I

Source Toxin synthesis Colony characters on BMAaCulture no. Geographical Substrate Ohran Citrinin Diameter tSeuxtrface Color Reverse Odor

NRRL 963 Washington, Air NDb ND Sc SFd FG 0' W'D.C.

NRRL 3586 Michigan Wheat flour ND ND S SF FG 0 WNRRL 3600 Pennsylvania Wheat ND ND S SF FG 0 WNRRL 5569 Kansas Corn ND ND S SF FG 0 WNRRL 5570 Unknown Oregano ND ND S SF GFGh GY' WA-14307 Michigan Wheat flour ND ND S SF FG 0 WA-15059 Wisconsin Corn ND ND S SF FG 0 WA-15105 Montana Wheat flour ND ND S SF FG 0 WA-15402 Texas Wheat flour ND ND S SF FG 0 WA-17919 Indiana Corn ND ND S SF FG 0 WA-18341 Kansas Corn ND ND S SF FG 0 WA-18343 Kansas Corn ND ND S SF FG 0 WA-18563 Michigan Wheat ND ND S SF FG 0 WA-18565 Michigan Wheat ND ND S SF FG 0 WA-18567 Michigan Wheat ND ND S SF FG 0 WA-18609 Michigan Wheat ND ND S SF FG 0 WA-18611 Michigan Wheat ND ND S SF FG 0 WA-18616 Michigan Wheat ND ND S SF FG 0 WA-18876 Michigan Wheat ND ND S SF FG 0 WA-19118 Michigan Wheat ND ND S SF FG 0 WA-19121 Michigan Wheat ND ND S SF FG 0 W

a BMA, Blakeslee's malt agar.bND, not detected.c Broadly spreading, rapid.dSlightly fasciculate marginally.' Forest green.' Orange.'Woodsy, earthy, penetrating.h Grayed forest green.'Golden yellow, diffusible.

two produced only citrinin and four producedonly ochratoxin.On Cz, colonies are 2.0 to 2.5 cm in diameter

in 12 days at 25 C, indistinctly zonate, consist-ing of a tough, somewhat raised and radiallywrinkled white to light cream mycelium; scantto abundant clear or faintly yellow exudate isproduced in small to conspicuous droplets onthe mycelium; poorly sporulating from the cen-ter outward; reverse colorless through variousyellow shades to brown; continuing to grow andreaching diameters of 4.5 to 5.0 cm at 1 month,in some strains remaining almost velvety but inthe majority becoming strongly fasciculate (Fig.2). Fasciculation, not evident in young cultures,appears to develop as a result of growth andregrowth over exudate droplets. Sporulation isheaviest in the fascicles which are formed inconcentric zones, leaving nonsporulating whitebasal mycelium exposed in alternate zones.Young conidia in both velvety and fasciculate

FIG. 1. Strain NRRL 963 representative of group IP. viridicatum on Cz (left) and BMA (right), 12 days,24 C.

strains are definitely blue-green but quicklychange to yellow-green and retain this color inage. Such a change was originally described forP. verrucosum Dierckx which was reduced tosynonymy with P. viridicatum by Raper andThom (13) and was cited as a characteristic of

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TABLE 2. Characteristics of P. viridicatum, group II

Source Toxin synthesis Colony characters on BMACulture no. Ochra- Surface

Geographical Substrate toxin Citrinin Diameter texture Color Reverse Odor

NRRL 3710a Canada Wheat + + Mb VC VGd PRBe P(?)fNRRL 3712 Canada Wheat + _- F" GG' UCJ P"NRRL 5571 Michigan Wheat + + R F GG PRB P(?)NRRL 5572 Canada Wheat + _ R F GG RB' PNRRL 5583 Canada Beans _ + R F GG PRB P(?)A-17732 Canada Wheat + + R F GG UC P(?)A-18686 Michigan Wheat + + R F GG PRB P(?)A-18689 Michigan Wheat + + R F GG PRB P(?)A-20216 Canada Beans + + R F GG RB P(?)A-20217 Canada Peanuts + _ R SF GG RB PA-20218 Canada Peanuts + + R F GG PRB P(?)A-20219 Canada Beans _ + M SF LGG" UC FW°A-20221a Canada Beans + + R F GG PRB P(?)A-20222 Canada Beans + + R F GG RB PA-20223 Canada Beans + + M FIP VLSq PRB P(?)A-20224 Canada Animal feed + + R F GG RB P(?)A-20225a Canada Peanuts + _ M SF GG UC P(?)

a Some color change on Czapek's solution agar or throwing sectors showing color change of group III.b Moderate growth, 3.0 to 3.5 cm in diameter at 2 weeks.c Velvety.d Yellow-green.e Pale reddish brown.'2-Pentanone?' Restricted growth, 2.0 to 2.5 cm in diameter at 2 weeks.hFasciculate.'Slightly grayed green.J Uncolored or light flesh.k2-Pentanone.'Red-brown.Slightly fasciculate.Light gray-green.

0 Faint woodsy.PFloccose.q Very lightly sporulating.

FIG. 2. Strain NRRL 5572 representative of group

HI P. viridicatum on Cz (left) and BMA (right), 12

days, 24 C.

P. palitans Westling strains used by Scott et al.(14).

Colonies on BMA are restricted, 2.0 to 2.5 cmin 12 days at 25 C, grayed yellow-green, granu-lar to definitely fasciculate marginally but, as

on Cz, continuing to grow and reaching adiameter of 3.5 to 4.0 cm at 1 month, becomingzonate and more conspicuously fasciculate dur-ing later stages of growth. Strains that remainalmoust velvety on Cz are more gray and lessfasciculate on BMA. Conidial chains in columnstend to form crusts; reverse ranges from uncol-ored to pale yellow to reddish-orange in ageshowing some red at colony centers. Penicillusmorphology is similar to that of strains in groupI (Fig. 3). Most produce a solvent odor, strong insome, barely detectable in others. The volatilematerial from NRRL 5572 has been identifiedby GLC and MS as 2-pentanonq.None of the cultures cited by Raper and

Thom as representative of P. viridicatum can beincluded in this group of isolates. They havebeen encountered most often on toxic grain fromCanada and Michigan. Incubation of isolationplates at 15 C, rather than 25 C, encouragestheir development.

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FIG. 3. Penicilli of strain NRRL 5583, representa-tive ofgroup HP. viridicatum, showing morphology ofstrains included in both groups I and II, x875.

Group III. Group III (Table 3) is a hetero-geneous assemblage of 13 strains isolated frommeat or air in a meat packing plant. Themajority were isolated quite recently from Euro-pean mold-fermented sausages and have a his-tory of variability in cultural pattern and coloras well as ability to produce ochratoxin. At one

time, all have produced the toxin; four strainshave lost this trait. None produced either cit-rinin or penicillic acid.

Colonies on Cz (Fig. 4) are close-textured,velvety, 3 to 3.5 cm in diameter in 12 days at25 C, plane to radially wrinkled, sporulatingabundantly in fairly bright yellow-green shadesbut quickly becoming various gray-brownshades; reverse uncolored to drab pink toorange-brown; exudate scant, occurring as very

small droplets within the basal mycelium; odorfaint, rather pleasant.

Colonies on BMA (Fig. 4) are plain, close-textured and velvety or showing an overgrowthof aerial mycelium to give a slightly floccosesurface; 3.0 to 3.5 cm in diameter in 12 days,reverse uncolored to light cream or dull paleyellow; odor faint, rather pleasant.

Penicilli of these isolates most frequentlyconsist of a terminal verticil of three or foursomewhat divaricate metulae 10 to 15 um long,

each bearing a verticil of fairly numerous paral-lel phialides averaging about 10,im in lengthwith distinct coidium-bearing tips 1 to 2 Mum long(Fig. 5). In more complex penicilli, branches arelong and only somewhat appressed and beareither a similar verticil of metulae and phialidesor phialides only. Conidia are mostly globose tosubglobose, smooth or nearly so, 3 to 4 gm inlonger axis, in occasional strains more definitelyelliptical but otherwise as above.

In growth habit on Cz, in the color change togray-brown and in the details of the penicilliand conidia, these strains satisfy Raper andThom's description of either P. olivino-virideBiourge or P. puberulum Bainier. Conidial coloron Cz is yellow-green as described for P. olivino-viride rather than the blue-green of P. puberu-lum. However, they have only a faint and notunpleasant odor on both Cz and BMA ascompared with the strong odors described forboth species, and they are unlike the type andrepresentative strains cited for either species.One culture (NRRL 1160) cited by Raper andThom as P. viridicatum, but not included inthis study, most nearly approximates theseisolates.

Despite certain cultural and morphologicaldifferences, the following two cultures havebeen included in group III because of theirsimilar origin, their lack of distinctive odors,and their toxin production pattern.

Strain NRRL 3711, yellow-green and in ageshowing only slight reduction in intensity ofcolor, has a history of having developed velvetysectors that showed the color change to brown.As it exists in our collection today, the cultureappears more funiculose than fasciculate on Cz.Penicilli and conidia are as described for thesausage isolates.

Strain NRRL 1161, cited as P. viridicatum byRaper and Thom, is velvety and definitelyblue-green when young on Cz but becomessomewhat fasciculate and assumes a yellowishtinge and remains green with age (1 month). Inthese characters, it resembles cultures of groupII to some degree. Colonies on BMA also resem-ble those of group II, but are somewhat morewidely spreading, considerably less fasciculate,and fail to produce the characteristic odor.Penicilli and conidia of this isolate also resem-ble those of group II.The considerable differences that separate

the isolates of group III from those of groups Iand II may mirror a selection effect of thesubstrate from which they were isolated, i.e.,low carbohydrate, high protein, and lipids (pri-marily saturated). This hypothesis will be sub-jected to future experimentation.

Odor. The solvent-like odor produced by

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TABLE 3. Characteristics of P. viridicatum, group III

Source Toxin synthesis Colony characters on BMA

Culture no. Substrate |Ohra- Citrinin Diameter Surface Color Reverse OdorGeographical Sbtae toxin CtinDamertexture Clr Rvre Oo

NRRL 1161 Canada Air in meat- + - Ma SFb G-BGc VCd FPepackingplant

NRRL 3711 Canada Ham + - M Fl' G-YG' UC FPNRRL 5573 Italy Sausage _ - M Vh G-YG UC FPNRRL 5574 Italy Sausage + - M V YG UC FPNRRL 5582 Italy Sausage + - M V YGi UC FPA-19166 Italy Sausage _ - M SF G-YG VC FPA-19169J Italy Sausage _ - M V-Fl G-YG UC FPA-19171 Italy Sausage + - M V YG UC FPA-19173} Italy Sausage + - M V YG UC FPA-19174 Italy Sausage + - M SF G-YG UC FPA-19175f Italy Sausage _ - M V YG UC FPA-19176J Italy Sausage + - M SF YG UC FPA-19179b Yugoslavia Sausage + M Fl NS" UC B'

a Moderate, 4.0 to 4.5 cm.b Slightly fasciculate.c Grayed blue-green.d Uncolored or in palest flesh tone.e Faint, pleasant.' Floccose.' Grayed yellow-green.h Velvety.I Yellow-green.J Unstable, throwing sectors of differing color or texture or both on BMA.A Nonsporulating.Butyric?

FIG. 4. Strain NRRL 5574 representative of themajority of strains in group III on Cz (left) and BMA(right), 12 days, 24 C.

NRRL 5572 (group II) or BMA was identified as

2-pentanone by GLC and MS (Fig. 6 and 7).Although the odor was not apparent when theculture was grown on barley or corn, it could bedetected by GLC. The odors of other strains ingroup II were determined organoleptically on

BMA. The strong woodsy or earthy odor ofgroup I gave no peaks on GLC, indicating a verylow concentration of what is probably a mixtureof volatile substances (10).Taxonomic implications. Exact delineation

of many species in the genus Penicillium is

extremely difficult. This is nowhere more ap-parent than in the Fasciculata section of theAsymmetrica.The Lanata, Funiculosa, and Fasciculata sec-

tions of the Asymmetrica are separated on thebasis of colony texture, as their names suggest.However, since determination of texture is nec-essarily subjective, sectional placement can bedifficult. Raper and Thom (13) were acutelyaware of the intergradations existing not onlybetween species but also between these sectionsand repeatedly advised users of their Manual toconsider species in more than one section.Those species in the section Lanata (lanoso-

viride, lanoso-coreruleum, biforme, commune,and lanoso-griseum) and the Funiculosa (psit-tacinum, terrestre, and solitum) that are distin-guished by subtle differences in conidial colorare those which show greatest resemblance toand are probably most often confused with thesimilarly separated P. viridicatum, P. cyclo-pium, and P. expansum series of the sectionFasciculata.

In most of the species concerned, morphologi-cal differences are essentially nonexistent. Peni-cilli are asymmetric, large, usually with one ormore appressed branches in addition to the

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new isolates from corn and wheat, there appearsto be a color continuum from the characteristicyellow-green of P. viridicatum to the distinctlyblue-green of P. martensii of the P. cyclopiumseries. A similar, but not entirely consistent,progression is observed in the amount and colorof diffusible pigment produced by these isolateson Cz slants: from yellow through orange andmaroon to purplish. Colony patterns are almostidentical. Differences in morphology are nebu-lous and a matter of degree rather than sharplydefined. The two cultures used to illustrate P.viridicatum (NRRL 963) and P. martensii(NRRL. 2029) in color plate VIII of the Manualof the Penicillia appear to represent the distalends of the continuum.Examination of the cultures cited by Raper

and Thom as representative of P. viridicatumreveals a considerable range of intraspeciesvariation. Their broad concept of the species isclearly apparent in their description. This con-

FIG. 5. Penicilli of strain NRRL 5582, also repre-sentative of group III, x875.

main axis, each terminated successively byveticils of metulae and phialides. Conidiophoresare comparatively long and coarse and may beeither smooth or rough. Conidia range fromglobose to elliptical, frequently in the samespecies, and are smooth or delicately rough-ened.Most species have been described as produc-

ing strong odors. Terms such as "actinomyces-like," "suggesting mushrooms," "moldy,""penetrating," "earthy," or occasionally "sour-ish" or "aromatic" have been used to distin-guish between these odors.

Representatives of many of the species as-signed to these sections show a marked tend-ency to vary under continued laboratory cul-ture. This variability, frequently reflected in theloss of a strain's ability to produce a specificsecondary metabolite, has plagued many inves-tigators. Whether such variation results from aninherent genetic instability, selection due tochange of substrate, the mutational effect oftoxic secondary metabolites, or other factors, isnot known.

Strains of Penicillium isolated from moldyfoods and feedstuffs encountered during myco-toxin investigations in recent years have blurredeven further the lines between species. Among

0 4 8 12 16 20 24Time Imoultesl

FIG. 6. Gas chromatogram of the volatile sub-stance produced by P. viridicatum NRRL 5572 grownon BMA.

100r

80

.60._

r-

'E 40

20

n

0

2 -Pentanone

I I I I I I-20 40 60 80 100 120

Mass NumberFIG. 7. Mass spectrogram of the volatile substance

produced by P. viridicatum NRRL 5572 grown onBMA. The mass chromatogram of pure 2-pentanoneis superimposible.

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cept is not contested and the fact that thecomposite of characters they used to delineatethe species was selected after years of experi-ence in the continued observation and cultiva-tion of many strains is fully recognized. Wecannot say, with certainty, that all of ourisolates would have been identified as P. viridi-catum by Raper and Thom. We believe thatthose separated into group I would definitelyhave been included. Those of group III mayrepresent either P. olivino-viride or P. puberu-lum, although they fail to conform with type orrepresentative strains of these species or withthe restrictedly growing strains of P. viridica-tum recognized by Raper and Thom. No coun-terpart for the isolates in group II has beenfound among type and representative strains ofeither P. viridicatum or those other species inthe sections Lanata, Funiculosa, and Fas-ciculata that Raper and Thom believed to showinterrelationship with P. viridicatum.The restricted growth on both Cz and BMA,

the distinctive odor, and the toxin productionpattern of this group of isolates perhaps wouldjustify their being described as a new subspeciesor variety. This is not being done, however,since they lack distinctive morphological char-acteristics; production of similar metabolicproducts does not provide an adequate basis forrecognition of a new taxon. In addition, GLCdetection of 2-pentanone in one of these isolatesdoes not constitute indisputable proof that theodor of the other isolates, organoleptically iden-tified as similar by several individuals, is due tothis same compound.

Until additional evidence is available, weprefer to apply the "group" system devised byRaper and Thom for P. citrinum, leaving all ofthe isolates in P. viridicatum but acknowledg-ing the existence of recognizable subgroupingswithin the species.The strains of P. viridicatum in this survey

can be separated into three groups according tothe following key: (1) Conidial color remainingyellow-green in age. (a) Colonies on BMAspreading, granular or slightly fasciculate atmargins in age .., group I. (b) Colonies onBMA restricted, usually conspicuously fascicu-late at margins in age ... group II. (1) Conidialcolor changing to gray-brown shades in age...group III.

ACKNOWLEDGMENT

We thank Robert Keliman for the mass spectral analyses.

LITERATURE CITED

1. Banks, G. T., K. W. Buck, E. B. Chain, J. E. Darbyshire,and F. Himmelweit. 1969. Virus-like particles in peni-cillin producing strains of Penicillium chrysogenum.Nature (London) 222:89.

2. Bozarth, R. F. 1972. Mycoviruses: a new dimension inmicrobiology, p. 23-39. Environ. Health Perspec. Issueno. 2, October, U.S. Dept. of Health, Education, andWelfare Publ. no. NIH 73-218. Washington, D.C.

3. Budiarso, I. T., W. W. Carlton, and J. F. Tuite. 1968.Hepatorenal damage in mice induced by Penicilliumviridicatum cultures, mycelia and chloroform extracts.Fed. Proc., Fed. Amer. Soc. Exp. Biol. 28:304.

4. Budiarso, I. T., W. W. Carlton, and J. F. Tuite. 1970.Phototoxic syndrome induced in mice by rice culture ofPenicillium viridicatum and exposure to sunlight.Pathol. Vet. 7:531-546.

5. Ciegler, A., Fennell, D. I., H.-J. Mintzlaff, and L.Leistner. 1972. Ochratoxin synthesis by Penicilliumspecies. Naturwissenschaften 59:365-366.

6. Ciegler, A., and C. P. Kurtzsman. 1970. Fluorodensitomet-ric assay of penicillic acid. J. Chromatogr. 51:511-516.

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