APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1983, p. 1311-13170099-2240/83/121311-07$02.00/0Copyright © 1983, American Society for Microbiology

Vol. 46, No. 6

Natural Occurrence of the Mycotoxin Viomellein in Barleyand the Associated Quinone-Producing PenicilliaBENEDICTE HALD,' DANIEL H. CHRISTENSEN,2 AND PALLE KROGH3*

Institute of Veterinary Microbiology and Hygiene, Royal Veterinary and Agricultural University,' ChemicalLaboratory V, the H. C. 0rsted Institute, University of Copenhagen,2 and Department of Microbiology,

Royal Dental College,3 Copenhagen, Denmark

Received 16 June 1983/Accepted 8 September 1983

In a batch of barley associated with field cases of mycotoxic porcine nephrop-athy and containing ochratoxin A and citrinin, the mycoflora were isolated byparallel incubation at 10 and 25°C. Subsequently, the isolated cultures werechecked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin,and citrinin). The nephrotoxin producers, all isolated by incubation at 10°C, werecomprised of one culture of Penicillium viridicatum, five cultures of Penicilliumcyclopium, and one culture of Penicillium crustosum, all producing xanthomegninand viomellein. One culture of P. cyclopium produced citrinin. Viomellein wasdetected in the barley at a concentration of approximately 1 mg/kg. The method ofanalysis for xanthomegnin and viomellein included extraction with chloroform,partitioning in hexane-acetone, and thin-layer chromatographic separation andidentification. The identity of the xanthomegnin and viomellein produced by theisolated fungi and of viomellein detected in the barley was supported by infraredspectroscopy. This is the first report of viomellein as a natural contaminant offoodstuffs.

Ochratoxin A is a major disease determinantof mycotoxic porcine nephropathy, an endemi-cally occurring disease in several Europeancountries (9). The kidneys, being the targetorgans, contain ochratoxin A, and renal analysisfor ochratoxin A residues has been extensivelyused in surveys for ochratoxin-associated por-cine nephropathy. Surveys in Denmark, Swe-den, and Hungary have revealed that 25 to 39%of swine kidneys with nephropathic lesions con-tain residues of ochratoxin A (8, 16, 17). Howev-er, the survey data also indicate that nephrotox-ins other than ochratoxin A might be involved inthe causation of mycotoxic porcine nephrop-athy, since only a fraction of the kidneys actual-ly contained ochratoxin. The fungal quinones,xanthomegnin and viomellein, might play acausal role in mycotoxic porcine nephropathy,although these metabolites so far have not beenencountered as natural contaminants of feed-stuffs. Renal lesions, comparable to ochratoxin-associated nephropathy, developed in swineduring ingestion offeed that was inoculated witha culture of Aspergillus ochraceus known toproduce xanthomegnin and viomellein (27).Here we report on the detection of viomelleinand the corresponding quinone-producing myco-flora in a barley batch which was associated withfield cases of mycotoxic porcine nephropathy.

(Part of this investigation was presented at the

Fifth International IUPAC Symposium on My-cotoxins and Phycotoxins, Vienna, Austria, 1 to3 September 1982.)

MATERIALS AND METHODSGrain sample. Since 1978, the Danish Veterinary

Service has been monitoring slaughtered pigs withnephropathic lesions for residues of ochratoxin A toprotect consumers against exposure to ochratoxin A(4). Farmers who have delivered pigs with ochratoxin-associated nephropathy have been advised to havetheir animal feed analyzed for ochratoxin A to improveanimal performance and the meat quality of baconpigs. Through this monitoring procedure, a sample ofbarley was obtained from a farm where recent cases ofporcine nephropathy had occurred. The sample con-tained ochratoxin and citrinin. Because of the pro-nounced mycotoxin contamination, the sample likelycontained other fungal metabolites, including qui-nones, and mycological and chemical investigationswere conducted as described in detail below.

Mycological investigation. Barley kernels were sur-face disinfected by treatment in 2% sodium hypochlo-rite for 1 min, followed by washing in sterile watertwice, and a total of 50 kernels were plated on maltagar (Difco Laboratories), with five kernels per petridish. Five petri dishes were incubated at 25°C for 2weeks, and five petri dishes were incubated at 10°C for3 to 4 weeks, followed by subcultivation on Difco maltagar (members of the order Mucorales) or on DifcoCzapek agar (Aspergillus sp. and Penicillium sp.) toobtain pure cultures. After identification to the genus

1311

1312 HALD, CHRISTENSEN, AND KROGH

level, the isolated cultures were tested for productionof nephrotoxins (ochratoxin, citrinin, xanthomegnin,and viomellein). Producers of nephrotoxins, all peni-cillia, were identified to the species level by the Raperand Thom classification described previously (13, 14).

Production of nephrotoxins. (i) Rice cultures. One-liter Erlenmeyer flasks containing 200 g of polishedrice and 100 ml of water were autoclaved at 121°C for45 min. The sterilized substrates were independentlyinoculated with spores of 21 isolated cultures as wellas spores of two known quinone producers, A. ochra-ceus M-298-SC and Penicillium viridicatum 66-88-2ATCC 36615 (supplied by J. Tuite, Purdue University,West Lafayette, Ind.). The flasks were incubated asstationary cultures at room temperature (18 to 20°C)for 21 days. After being treated with 1% propionic acidto kill the fungal organism, the cultures were dried at40°C for 5 days (15).

(ii) Fungal mats. Fungal mats were obtained fromselected isolated cultures and from a known quinoneproducer, A. ochraceus M-298-SC, and a known och-ratoxin and citrinin producer, P. viridicatum 854.Cultures were grown in 450-ml Duran flasks containing100 ml of Czapek Dox broth (Difco) containing 1%corn steep liquor adjusted to pH 7.0. The medium wasautoclaved at 121°C for 15 min, inoculated with fungalspores, and incubated as stationary cultures at roomtemperature (10 to 16°C) for 16 days. After removal ofthe fungal mats from the broth, they were coveredwith chloroform for 3 days to kill the fungal organismand dried on filter paper at room temperature for 3days.Chemical analysis for nephrotoxins. (i) Barley and

rice. The concentration of ochratoxin A and citrinin inthe barley sample and the rice cultures was measuredby thin-layer chromatographic procedures, includingconfirmation tests for identification of ochratoxin A(10) and citrinin (6). The quinones were detected andquantitated by a thin-layer chromatographic proce-dure, which was modified from three published proce-dures (3, 15, 21). The procedure involved extraction ofa finely ground, 50-g sample with 250 ml of chloroformin a Waring blender for 5 min. After filtration, theextract was evaporated to dryness, and partitioning ofthe residue was performed in 100 ml of n-hexane-90oacetone in water (1:1). The acetone layer was collect-ed, and partitioning of the n-hexane layer was repeat-ed. The combined acetone layers were extracted twicewith 50 ml of chloroform, and the combined chloro-form extract was dried over anhydrous sodium sulfateand evaporated to dryness.

(ii) Fungal mat. The fungal mat was defatted in n-hexane for 24 h. After the hexane was discarded, 5 mlof 1 N hydrochloric acid was added, and the mat wasextracted twice with 100 ml of chloroform. Afterfiltration, the combined chloroform portions weredryed over anhydrous sodium sulfate and evaporatedto dryness.

Thin-layer chromatography was performed on silicagel (60 HR; E. Merck A.G., Darmstadt, FederalRepublic of Germany) in an unequilibrated, unlinedtank with toluene-ethyl acetate-formic acid (6:3:1) for40 min. After drying, the plates were observed underlongwave UV light and daylight. The plates wereexposed to ammonia fumes and examined again underlongwave UV light and daylight. Xanthomegnin (Rf,0.45) and viomellein (Rf, 0.59) were identified accord-

ing to their positions on the plate and their color spots.Quantitation was made by comparing color intensitiesof xanthomegnin or viomellein spots, or both, of thesamples with those of standard spots. Standard solu-tions were made daily because the compounds areunstable in solution (24); the concentrations of thestandard solutions were checked by UV spectrometryat 264 nm (e = 19,300 for xanthomegnin; e = 20,200 forviomellein). The precision of the procedure was deter-mined by analysis of 10 identical samples of A. ochra-ceus-inoculated rice. The sensitivity and accuracy wasdetermined by recovery studies on barley spiked withvarious amounts of crystalline xanthomegnin and vio-mellein.

Confirmation of quinone identity by infrared spec-troscopy. The fungal quinones were isolated by prepar-ative thin-layer chromatography on silica gel plates(500 jm thick) with toluene-ethyl acetate-formic acid(6:3:1) as the developing solvent. The xanthomegnin-and viomellein-containing bands were scraped fromthe plates and extracted by shaking for 20 min on alaboratory mixer three successive times with chloro-form to which had been added a few drops of formicacid. The xanthomegnin- and viomellein-containingextracts were rechromatographed on preparative silicagel plates and developed with toluene-ethyl acetate-formic acid (6:3:1) to eliminate contaminating com-pounds from the quinones (15). Authentic samples ofxanthomegnin (provided by R. Peterson, NorthernRegional Research Center, Peoria, Ill.) and viomellein(supplied by M. E. Stack, Food and Drug Administra-tion, Washington, D.C.) were similarly subjected topreparative thin-layer chromatography to eliminatepossible oxidative degradation products incurred dur-ing shipment and storage. The infrared (IR) absorptionspectra were obtained directly on the chloroformextracts with 0.2- or 0.5-mm liquid cells with KBrwindows. The transfer of the solutions to the cells wasperformed quickly and with minimal exposure to air,and the spectrum was run immediately. The IR spectrawere recorded and the data stored digitally with aPerkin-Elmer 580 IR spectrometer interfaced with anRC 4000 computer (Ballerup, Denmark). Since theamounts of formic acid present in the chloroformsolutions varied considerably and gave rise to strongbands, a direct compensation of both chloroform andformic acid could not be performed with the referencebeam of the spectrometer. Instead, the compensationwas made digitally by subtracting chloroform plusformic acid and pure chloroform in such a ratio thatboth the formic acid bands and the chloroform bandsdisappeared from the spectrum. This technique is notfully satisfactory since parts of the spectrum may bepoorly recorded. The use of the KBr disk technique,combined with an evaporation of the solvent on theKBr powder, was tried but failed to give reproduciblespectra due to chemical changes during the samplepreparation.

RESULTSAnalysis of the precision of the thin-layer

chromatographic procedure gave the followingresults: xanthomegnin, average, 21.1 mg/kg; co-efficient of variation, 5.5%; viomellein, average,77.9 mg/kg; coefficient of variation, 2.6%. Therecovery study revealed an average recovery of

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MYCOTOXIN VIOMELLEIN IN BARLEY 1313

TABLE 1. Recovery of xanthomegnviomellein from barley

Quinone added (mg/kg)a

Xanthomegnin0.500.50

1.001.002.502.505.405.4010.0010.0027.0027.00

Viomellein0.500.501.001.002.502.505.005.0010.0010.0083.0083.00

a For xanthomegnin, the average is 45.coefficient of variation is 19.1%; for vioIaverage is 58.8% and the coefficient of16.0%o.

b ND, Compound not detected.

45.4% for xanthomegnin and 58.8% flein, with a lower level of detection(Table 1).

in and The mycoflora isolated from the barley byincubation at 25°C included four cultures of

% Recovery Aspergillus sp., two cultures of Penicillium sp.,and two cultures belonging to the order Mucor-

NDb ales; none of the cultures produced any of theND nephrotoxins analyzed for (ochratoxin, citrinin,53 xanthomegnin, viomellein). The mycoflora ob-62 tained from low-temperature incubation (10°C)32 consisted of 21 cultures of Penicillium sp., and52 eight (38%) of them produced nephrotoxins un-43 der laboratory conditions. Seven cultures pro-37 duced xanthomegnin and viomellein, as mea-49 sured by thin-layer chromatography, and one46 produced citrinin (Table 2). No ochratoxin pro-46 ducer was isolated. The IR spectra comparing

xanthomegnin and viomellein isolated from P.viridicatum 852 (the main producer of quinones

ND from the barley sample) with reference com-ND pounds, as well as with xanthomegnin and vio-74 mellein isolated from A. ochraceus M-298-SC50 (the known quinone producer), are shown in60 Fig. 1. The spectrum of reference xanthomegnin75 was obtained from a solution of 1 mg of xantho-54 megnin in 0.3 ml of pure chloroform, whereas53

the spectra of the two fungal cultures, P. viridi-46 catum and A. ochraceus, were measured with57 the extract described above. Consequently,59 there are some minor differences between the

4% and the spectra due to the incomplete compensation ofmellein, the the chloroform and formic acid bands in thevariation is spectra of the extracts. The spectrum of xantho-

megnin isolated from P. viridicatum 852 is es-sentially identical to the spectra of referencexanthomegnin and of xanthomegnin isolatedfrom A. ochraceus.

or viomel- The spectrum of reference viomellein, howev-at 1 mg/kg er, was measured on a chloroform-formic acid

extract. Consequently, interference from formic

TABLE 2. Thin-layer chromatographic analysis of rice cultures and fungal mats of Penicillium culturesisolated from a barley sample naturally contaminated with viomellein, ochratoxin A, and citrinin

Rice Fungal matSpecies Culture no. IMIa ATCCb Xanthomegnin Viomellein Xanthomegnin Viomellein

(mg/kg) (mg/kg) (mg/mat) (mg/mat)

P. viridicatum 852 2652% 48413 11.3 31.3 9.6 7.2P. cyclopiumc 853 265297 0 0 0 0P. crustosum 884 265298 48414 0.3 0.4 0.2 0.8P. cyclopium 885 265299 48415 0.3 0.1 6.0 0.9P. cyclopium 886 265300 48416 Tr Tr 0.8 0.9P. cyclopium 887 265301 48417 1.5 6.3 1.2 1.8P. cyclopium 888 265302 48418 Tr Tr 0.3 0.2P. cyclopium 889 265303 48419 0.2 0.8 12.0 3.6A. ochraceusd M-298-SC 187.5 62.5 8.0 4.8P. viridicatumd 66-68-2 36615 21.0 2.0P. viridicatume 854 0 0 0 0

a Accession number, Commonwealth Mycological Institute, London.b Accession number, American Type Culture Collection, Rockville, Md.c Rice culture contained 1 mg of citrinin per kg.d Reference cultures (quinone producers).' Reference culture; fungal mat contained 4 mg of ochratoxin A and 1 mg of citrinin.

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1314 HALD, CHRISTENSEN, AND KROGH

T

T

-0 a _w am 11a 1ismlw mFIG. 1. Comparison of IR spectra of xanthomegnin and viomellein isolated from P. viridicatum 852 with

those of reference compounds and of xanthomegnin and viomellein isolated from A. ochraceus M-298-SC, aknown quinone producer. (A) Reference xanthomegnin; (B) xanthomegnin from P. viridicatum; (C) xanthomeg-nin from A. ochraceus; (D) reference viomellein; (E) viomellein from P. viridicatum; (F) viomellein from A.ochraceus. Abscissa, wave number (cm-'); ordinate, percent transmission (%T).

acid bands occurred in all three spectra of vio-mellein. However, by examining the region withminor interference (1,800 to 1,250 cm-'), thesame bands can be recognized in all three spec-tra. Strong interference was observed in thefollowing regions: 3,700 to 3,400 cm-'; 3,100 to2,900 cm-'; 1,250 to 1,050 cm-'; 850 to 600cm-1. In spite of this interference, the identity of

the viomellein spectrum from P. viridicatum 852with the spectra of reference viomellein and ofviomellein from A. ochraceus is reasonably wellensured. The identity of citrinin from Penicilli-um cyclopium 853 was established by formationof the acetate derivative (6). This is the firstreport of P. cyclopium as a citrinin producer.

All eight toxin producers were terverticillate

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MYCOTOXIN VIOMELLEIN IN BARLEY 1315

S vX ~~~~"t.,X5--==; w7;.

FIG. 2. P. viridicatum 852 (left) and P. viridicatumNRRL 963, the type culture described previously (16)(right), were incubated on malt agar for 3 weeks at25°C. Note the fasciculation in the marginal area ofculture 852 (left).

organisms with rough stipes, showing fascicula-tion at the marginal area of the colonies (Fig. 2).The main quinone producer, P. viridicatum 852,formed granular colonies on Czapek agar, attain-ing a diameter of 3.6 to 4.3 cm in 2 weeks at25°C, which is comparable with the type culture,P. viridicatum NRRL 963, described by Raperand Thom (14). The conidial area was yellow-green and remained so with age. On malt agar,growth of P. viridicatum 852 was restricted,

compared with the type culture, P. viridicatumNRRL 963 (Fig. 2). The six P. cyclopium cul-tures (853, 885, 886, 887, 888, and 889) hadcolonies with spreading growth on both Czapekand malt agar, with blue-green conidial areas onboth media. Culture 884, Penicillium crustosum,grew as a spreading colony on Czapek and maltagar, with yellow-green conidia that formedcrusts. The culture produced a limited rot ofpomaceous fruits. This is the first report of P.crustosum as a quinone producer.

Thin-layer chromatographic analysis of thebarley sample demonstrated the presence of 1mg of viomellein per kg, in addition to 1.9 mg ofochratoxin A per kg and 0.8 mg of citrinin perkg. The IR spectrum of the extract from thebarley sample shows that viomellein is contam-inated with a compound having a strong band at1,750 cm-' (Fig. 3). However, the bands ofviomellein from the barley sample in the regionfrom 1,800 to 1,300 cm-' can be distinctlycompared with those of reference viomellein andof viomellein from A. ochraceus. Outside thisregion it is difficult to distinguish viomelleinbands in the spectrum from the barley samplebecause of the interference from formic acidbands. In conclusion, there are several indica-tions in the IR spectrum of the presence ofviomellein in the barley sample.

DISCUSSIONBy comparison of the IR spectra of xantho-

megnin and viomellein from P. viridicatum 852with published IR spectra (15) and data from IR

4000 MM mm0 20 WO 150 1000 500

FIG. 3. Comparison of IR spectrum of viomellein isolated from a barley sample with those of the referencecompound and of viomellein isolated from A. ochraceus M-298-SC, a known quinone producer. (A) Viomelleinfrom A. ochraceus; (B) reference viomellein; (C) viomellein from barley.

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1316 HALD, CHRISTENSEN, AND KROGH

spectroscopy (22) of these compounds, a satis-factory agreement is evident. The viomellein IRspectra of Robbers et al. (15) show a strong bandat 1,625 cm-1, which is not present in ourspectra (Fig. 1). Generally, the minor differencesobserved between the various IR spectra ofviomellein are most likely due to small amountsof degradation products incurred during samplestorage and handling.The IR spectrum of viomellein from the barley

sample provides only a limited degree of identifi-cation per se because of the presence of acontaminating compound as well as interferencefrom the solvent used in isolation of viomellein(Fig. 3). However, the pronounced fungal poten-tial for quinone production, demonstrated by theisolation of seven quinone-producing culturesbelonging to three species of Penicillium (Table2), provides convincing evidence that the isolat-ed compound from barley is viomellein. This isfurther supported by the performance of thecompound during thin-layer chromatography.The quinone pigment xanthomegnin was first

discovered in cultures of the dermatophytesTrichophyton megnini and Trichophyton rubrum(7, 24, 25). Xanthomegnin and viomellein havesubsequently been identified in cultures of otherdermatophytes, as well as in cultures of speciesbelonging to Aspergillus and Penicillium (Table

TABLE 3. Fungal producers of quinones(xanthomegnin and viomellein)

Fungal organism Xantho- Vio- Refer-megnin mellein ence

DermatophytesT. megnini + 7T. rubrum + 24, 25Trichophyton + 11violaceum

Microsporum cookei + 12Nannizzia cajetani + + 19

(perfect state ofM. cookei)

A. ochraceus groupAspergillus sul- + + 5phureus

Aspergillus + + 15auricomus

Aspergillus melleus + + 5, 15A. ochraceus + + 15, 21Aspergillus ostianus + + 15

PenicilliaP. viridicatum + + 3, 15,

21, 22P. cyclopium + + 21P. crustosum + + This

report

3). The concentrations reported for xanthomeg-nin and viomellein in fungal cultures vary con-siderably, even for the same culture, and levelsmore than 100 times higher than those indicatedin Table 2 have been observed. It should, how-ever, be kept in mind that all published methodsof analysis for the quinones are highly inade-quate in terms of sensitivity, and optimal condi-tions for fungal production of quinones are rudi-mentarily elucidated for the same reason.

It is noteworthy that all toxin-producing Peni-cillium cultures were isolated at a low tempera-ture of incubation (10°C), a feature cortpborat-ing earlier observations of ochratoxin A- andcitrinin-producing penicillia being isolated at10°C (18). In identifying the isolated penicillia,the taxonomic classification described previous-ly (14, 16) was employed, in which P. viridica-tum and P. cyclopium are distinct species, al-though they are closely related.

Ciegler et al. (2, 3) divided P. viridicatum intothree subgroups, of which group II comprisedochratoxin and citrinin producers and group Iincluded xanthomegnin and viomellein produc-ers. The quinone-producing culture of P. viridi-catum 852 in this study is an intermediate be-tween groups I and II, as it grows in a spreadingmanner on Czapek agar (group I) and in arestricted manner on malt agar (group II).No ochratoxin producer was isolated from the

barley sample, probably because the responsibleorganism was no longer viable or because theorganism had been so unevenly distributed inthe barley that 25 kernels per level of incubationtemperature did not provide a representativesample for purposes of isolation. However, thisobservation is not entirely unusual, since noochratoxin producer was isolated from the cornsample when this compound was first encoun-tered as a natural contaminant (20).

Crystalline xanthomegnin and viomelleinwere fed separately to two groups of mice duringa 10-day trial (1). They developed identical le-sions, predominantly in the liver, including nec-rotizing cholangitis, focal hepatic necrosis, andhyperplasia of the biliary epithelium, with onlyminor changes in the kidneys; this spectrum oflesions was previously observed in rodents thatwere fed cultures of quinone-producing P. viridi-catum and other species. Quite oppositely, thekidneys were the target organ in pigs fed a rationcontaining a rice culture of A. ochraceus M-298-SC (a known quinone producer) during a 2-month trial (26, 27). The renal lesions developedincluded tubular necrosis with thickened base-ment membranes and cortical fibrosis, which iscomparable with ochratoxin A-associated por-cine nephropathy (9).The modified thin-layer chromatographic

method of analysis used in this study has better

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MYCOTOXIN VIOMELLEIN IN BARLEY 1317

recovery (Table 1) than a previously reportedhigh-pressure liquid chromatographic procedure(21). However, the sensitivity is poor, with alimit of detection at about 1 mg/kg. Since thefungal potential for quinone production is high,with 11 to 33% of the more common penicillia ingrain, P. cyclopium and P. viridicatum, able toproduce xanthomegnin or viomellein or both(23), the inadequate chemical methods of analy-sis appear to prevent the elucidation of the truefrequency and levels of quinone contaminationof foodstuffs.

ACKNOWLEDGMENTSThe valuable assistance of A. H. C. Onions, Common-

wealth Mycological Institute, Kew, England, and of A. Mad-sen, National Institute of Animal Science, Copenhagen, Den-mark, is acknowledged.

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26. Zimmermann, J. L., W. W. Carlton, and J. Tuite. 1979.Mycotoxicosis produced in swine by cultural products ofan isolate of Aspergillus ochraceus. I. Clinical observa-tions and pathology. Vet. Pathol. 16:583-592.

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VOL. 46, 1983