3678 SYNERGISTIC COLOR Varlants of AUREOBASIDIUM

3678

p Reprinted from }yfyroLOGIA, Vol. LXVII. No.2, {)p. 342-361, Mar.-Apr., 1975 Printed in U. S. A.

SYNERGISTIC COLOR VARlANTS OF AUREOBASIDIUM PULLULANS

L J. WICKERHAM AND C. P. KURTZMAN Northern Regional Research Laboratory, Ag~iturai Research Seruice, U. S. DepQTtment of Agriculture, Peoria, Illinois 61604

SUMMARY Color variants of Aureobasiditlm puliulans were isolated from mao terials collected in tropical and subtropical regions of the world. Three different types of variants produce colonies that are either yellow, red. or purple. When the variants are either mixed with or grown near many different species of yeasts and other microfungi. the rapidity and intensity with which pigment is produced are markedly enhanced. Yel low variants are moderately unstable and change to red. The red varia· ants are highly unstable, changing to yellow. Reversion to the normal wild type is seldom observed. Addition of acid or base causes the pigments to function as pH indicators. Pigment formation is tempera· ture sensitive and mixtures of variants and elicitor strains are colorless at 35 C. but show typical enhancement of pigmentation when removed to 25 C. Variants incubated alone at 35 C show temporary intense pigmentation upon removal to 25 C.

Atereobasidium puliulans (DeBary) Arnaud. known also as Ptel. lularia pullttlans (DeBary) Berkhout. is one of the commonest of the microfungi. Cooke (1959, 1962) and Cooke and Matsuura (1963) pointed out that this species is extremely variable and presented argu ments for the name Aureobasidittm pullulans. Its ability to produce black pigment, characterized as melanin by Lingappa et al. (1963), is well known. That it occasionally produces intense pigmentation of other colors is less well known, although Lodder (1970b) mentions the ability of A. pullulans to produce a deep orange color. During the past 16 yr, variants of this species were isolated from nature that produced a variety of colors, and growth of the variants with many other micro fungi caused a marked increase in the rapidity and amounts of the pigments produced. This report concerns these natural color variants of A. pullulans and some of the factors influencing pigment formation.

MATERIALS AND METHODS The three media used most commonly in this study were yeast e."{ tract-malt extract medium (YM), malt e.,,<:traet medium (ME), and 342 Purchased by Agricultural Research Service U. S. Department of Agriculture For Official Use 343 MVCOLOGIA, VOL. 6i, 19i5 yeast nitrogen base (YNB). All were previously described (Wicker ham, 1951; Anon., 1953). The respective pH of these media are 6.5, 5.2, and 5.6. Color variants were isolated from nature on isolation medium (lM) and 20% glucose medium (20D) ("Wickerham, 1969). The identification procedures used were described by Wickerham ( 1951 ). Ta.'Conomy and nomenclature accepted by the various authors of The Yeasts. edited by Ladder (1970a), are followed for the yeasts except for the genus Saccharomycopsis (van der \Valt.and Scott. 1971). and the paper by Arnold and Ahearn (1972) is followed for the genus Prototheca. Inocula were prepared as follows for testing the ability of strains to act as elicitors on solid media: Both color variants and elicitors were grown separately on Y)''I slants for 24 or 48 hr at 2S or 28 C. Then approximately one volume of cells of the color variant and two-thirds volume of elicitor cells were mixed with a loop near the edge of a y)r agar plate. A tiny amount of the mixture was touched first to the surface of the agar a few millimeters away by needle and then streaked by loop across the remaining sterile ~o-ar on the plate. For tests using slants, the mixtures were made directly on the slant and sprend over the sur face of the agar. Controls of each culture separately were streaked on plates or slants. lncubntion was at 2S or 28 C. and tests were read at either 24 or 48 hr. or both. and sometimes agnin after several days. Inocula for shaken flasks were grown on YM slants. and the entire growth from a 24-hr slant wa$ removed to a test tube containing 4.5 ml of sterile distilled water. An approximately equal cell density was achieved for all strains tested by use of a photometer or a carn with inked lines. The inocula of the elicitor strains were made up to about two thirds of the density of the color variant inoculum. Each test flask received 0.1 ml each of color variant and elicitor. and each control received 0.2 ml of the single culture with which it was inoculated. The cultures were usually shaken on a reciprocal shaker but sometimes on a Gump rotary shaker. and incubation was generally at 25 C but occasionally at 28 C as gh'en in the text.

SOURCES AND DESCRIPTIONS OF CULTURES Cultures from soil. Puerto Rico.-On April 13. 1956, Dr. Richard VV. Jackson, retired from the Northern Laboratory. collected a soil sample from the lawn of the Experiment Station at San Juan. After enrich ment of the soil sample by two serial transfers in shaken 200 isolation medium at 28 C followed by development on streaked YM agar plates. vVrCKERHAM AND KURTZ:-'IAN: ACREOBASIDruM Pl'LLUL.\NS 3-l:4 only colonies of yeasts and yeastlike organisms were obtained. One type of isolate, designated NRRL YB-4026, appeared to be a variant of A. pttllulans. Isolated colonies when 1-2 da old were light yellow. glistening, butyrous, and filamentous, becoming pink within a few days. Typical white mucoid colonies of A. pullulans (strain NRRL YB-4029) were present, which became butyrous and black with age. Usually colonies of A. pttllulans on Y"N[ agar are pale pink, white. gray. or black when young, with an early mucoid consistency which later be comes butyrous. Hyphae develop around the edges of the colonies. especially where crowded. Also on the isolation plates were two types of Rhodotorula colonies, NRRL YB-+027 R. gill tinis (Fres.) Har rison Val'. glutinis which were less mucoid than colonies of the second type, NRRL YB-+028. The remainder of the colonies on the plates were of a white yeast strain NRRL YB-+030 Candida !Juilliermondii (Castellani) Langeron et Guerra VaT. guilliermondii. 'When the isolation plates were only 18 hr old. colonies of the ap parent variant of A. pullulans were yellow where well isolated from. light red where rather near, and blood red where close to the Rhodo torula colonies. Cells of the deeply red colonies of YB-4026 streaked on YM agar in pure culture did not reproduce the intense piRmentation that proximity to the Rhodotorula had elicited. Mixtures of YB-I-026 with the two isolates of Rhodotorula and one isolate of C. gllillier mondii val'. guilliermondii reproduced the synergistic production of pigment. The less mucoid Rlzodotorula strain YB-I-027 was a stronger elicitor than the more mucoid YB-4028. Candida guilliermolldii var. guilliermondii YB-4030 was also inferior to YB--l-027. A mixture of YB-4026 and typical A. pullulans YB-4029 were not synergistic. nor 'was YB-4029 with YB-4027 or YB-4030. Triplicate colonies ofcolor variant YB-4026 growing on Y)'[ agar in petri dishes frequently produced more rapidly growing orange sec tors. Cells from such outgrowths were synergistic in their produc tion of a deep red pigment with elicitor strains. A large sector was streal

red and yellow variants with more intense synergistic colors than YB-4D26.

Cultures from slash pine, Sebring, Florida.-In June 1959, one of us (L. J. W.) collected at Highlands Hammock State Park in south central Florida a sample of insect frass mixed with pitch that occurred close to an old slash made on a live, large slash pine [Pinus elliottii Engelm. val'. elliottii (Little, 1953)] for collecting pitch used in the production of naval stores. Enrichment was bv two serial transfers in shaken flasks containing half Y?vI broth and half IM. Colonies on the isolation plates were of two types of A. pltllulans, and apparently the few other colonies of microorganisms that were pres ent were incapable of acting as elicitors. Hundreds of the colonies were nearly white. almost without hyphae, but with a Slight suggestion of red pigment. Eight were deep red. One of the nearly white colonies was streaked on Y:YI plates, and the colonies produced after i da of growth were similar to those formed by cells of the red colony except for pigmentation. The colonies developed a weak orange color and small dark centers. The culture was lyophilized as )l'RRL YB-458i. One of the strongly pigmented colonies was streaked on Y)'{ plates. The colonies that developed were uniform where moderately well iso la.ted. When i da old, these colonies had an outer red zone. a slightly yellowish central zone. and a gray center. TIle cells were typical of A. pullulans, including immature unseptate chlamydospores. The cul ture was lyophilized as NRRL YB-4588. Cultures of YB-4587 on slants at i cia were pink on Y?\,( and black on ME agar, the growth on the latter showing black and brown septate spores and hyphae; no free oil globules were observed. Strain YB 4588 on YM slants of the same age had yellow central growth and were red at the edges; on :-.[E slants the ba:;e was yellow and the top was reddish. Hyphae had few septa, the cells were yellowish or slightly brown, and many free large oil drops were present. Isolate YB-4587 did not grow at 36 C on YM plates. but YB-4588 grew slightly at this temperature, growth being limited almost exclu sively to fission of spheroidal cells. One plate of each culture was taken from 36 C and placed at 25 C. Isolate YB-4587 showed no growth. indicating it had been killed by the high temperature. but YB-4588 de veloped colonies that had a broad, dark red zone, a grayish middle zone. and a tiny black center. The dark red colony form was assumed to be synergistic, but synergism was not tested previous to lyophilization. When the lyophilized cultures of these two strains were revived in VVICKERHAM AND KURTZMAN: AUREOBASIDIUM PULLULANS 346

1970, the colonies were typical of A. pullulans,o neither developed red or orange color, nor were they synergistic. Strain NRRL Y-6754a from tropical foliage.-On November 10, 1964, Dr. M. H. Deinema sent cultures isolated by Dr. J. Ruinen from trop ical foliage in Java and Surinam (Ruinen, 1963; Ruinen and Deinema, 1964). Among them was culture 272 (NRRL Y-6754a) labelled A. pullulans. This culture produced on Y?vI triplicate colony plates fur rowed colonies that at 6 da were colorless but were pale pink at 15 da; the colonies on l'vIE plates were furrowed and pale purple at 6 da, be coming entirely filamentous, glistening, and surrounded by black hyphae. \Vhen 20 da old, however, an outer zone was chocolate brown to black. The black hyphae had dark brown hypha! cells and chlamydospores; some of the latter were septate. Strong synergistic purple pigment was produced with elicitor YB....j.()27 on a YM slant at 24 hr, but synergistic action in liquid media was scant. Cultures from slash pine, Fort Meyers, Florida.-Strain Y-2311 was isolated from frass collected by the late Dr. Ernst A. Bessey on De cember 30, 1950, from a decaying stump of slash pine located in the vicinity of Fort Meyers, Florida. The yeast was isolated following en riclunent in 1M. Many colonies on the Y),I isolation plates when 4 da old were intense red and butyrous. with long hyphae (strain Y-2311). Similar colonies were gray with red sectors, others were exclusively gray. A number of other colonies showing varying degrees of resem blance typical of A. p1tllulans were present. Strain Y-2311 was transferred to YM slants and to slants of a similar medium that contained 2% sucrose instead of the 1'70 glucose in Y?vI medium, and no malt extract. The Y:'I slant cultures of Y-2311 were pink at 2 and 3 da. The surcose slant culhlres were yellow at 2 da, but at 3 da the top half was red and the bottom half was yellow. In July, 1960, the red strain Y-2311 (henceforth referred to as the parent red culture) was inoculated onto the surface of Y),'f agar solidi fied in a vertical test tube. As soon as growth had de\'eloped and the culture was still pale, it was covered with sterile mineral oil to a depth of 1 em and stored in an upright position at 8 C. The oiled cul ture was uniformly black and tough when cultured in July, 1964. Cells from the black growth were streaked on YiYr agar where they produced red to nearly white growths. Cells were streaked on :'IE and Y)'1 plates. Eight days later the ME plates streaked from a red area had colonies about two thirds of which were a rich golden yel low (Y-2311-3): the remainder were red (Y-2311-2). All the red 347 MYCOLOGIA, VOL. 67, 1975

colonies had developed. black centers; the yellow colonies had not. Cells from a red area produced e..'Cclusively red colonies on YM plates and none of them had black centers. The red colonies on ME were a darker, less brilliant red than the colonies on Yl'1 plates. Two color types of variants were present. The red type was red on both media and sufficiently stimulated by ME agar but not by YM agar to produce dark mycelium and chlamydospores in the center of the colony at 8 da. The yellow type was yellow on ME but red without black centers on Yl'I. Both color variants were red on Y?vI streak plates and neither produced. black mycelium. Cells streaked from a nearly white area yielded. pale pink colonies. One was designated as variant Y-2311-1. It evidently was a revertant to the normal form of A. pullulans. The variants were monitored by their growth on ME and YM streak plates, on ME slants, or as triplicate colonies on 1'1E plates. Streak-plate cultures on both med.ia are recommended. Lower temperatures, to 8 C, longer incubation, to several weeks, and :'vIE medium are factors favoring the development of black color in A. ptel lulans. As is common for newly isolated. A. pttllttlans, colonies on streak plates of all three variants were more or less mucoid when 1-2 da old and became less mucoid with age. The mucoid character was generally stronger on 1'IE than on Y?v1 agar. The colonies of variant -1 were pink on both media and did not change color except for the develop ment of black hyphae on old ME cultures. The colonies of -2 grew more slowly and produced. relatively more hyphae than the other variants; they became red on YM agar in 4 or 5 da. Entire colonies were bright red. where crowded.; lighter areas became apparent in well separated colonies. Colonies of -2 on ME plates were red, becoming black when 8 or 9 da old; the peripheral colonies remained red some what longer than the central colonies. Variant -3 produced on Y1'1 streak plates colonies that were yellow when young and later changed to pink. Colonies on ME plates were yellow, with occasional bright red sectors. Cultures on ME slants were more distinctive than cultures on YM slants, distinction between variants having occurred when the cultures were about 4 da old. On YM slants from 5 to 15 da of age, variants -1 and -3 were pale pink and -2 was red. On ME slants at 4 to 5 da, variant -1 was pale pink, -2 was red, and -3 was yellow. At 15 da, variant -1 remained unchanged. The growth of -2 had changed. to dark brown-red on the upper half of the slant and black on the lower half with intense red-brown pigment throughout the agar. VVICKERHAM AND KURTnfAN: AUREOBASIDIUM PULLULANS 348

The three variants of strains Y-2311 were inoculated onto YM and ME agar plates as triplicate giant colonies to supply more complete descriptions than just given for the slant cultures and for possible indi cations of frequency of variant formation. The YM plate cultures showed no soluble pigment and were outstanding for Y-2311-2, which consistently produced colonies with characteristic brilliant red triangular rays against a pale red background of diverging rays e.'(tending almost to the edge of the colonies. The following descriptions report changes that occurred during 23 da of incubation of triplicate colonies on ivIE agar plates. Variant Y-2311-1 showed the least change; it was pale pink through out 23 da of incubation. Variant Y-2311-2 at 7 da had a cream center; a broad, highly glistening, leathery textured, orange-red, radially furrowed middle zone, and a slightly orange-red hypha! zone. Intense red soluble pig ment occurred in the agar. At 15 da the centers were convex, yellow, and mucoid; then a reddish-black, furrowed, glistening, middle zone gave rise to hyphae which in the proximal part of their zone were red. then changed to yellow, and finally to gray. At 23 da the entire colonies were glistening, and the centers were no longer mucoid; they were black except for a narrow gray edge, and the agar remained red with soluble pigment. The triplicate colonies of Y-2311-3 at 7 da had small black centers; the black color radiated like hyphae into a golden yellow outer zone. At IS da the centers were blqck surrounded by a yellowish zone, both zones highly glistening and submucoid ; succeeding zones were (1) orange with irregularly circular folds, (2) broad yellow butyrous, (3) orange edge, and (4) pale yellow and gray hyphal zone. At 23 da the colonies were yellow with red edges, and one had a large black sector. Scant purple pigment apreared in the agar. Although triplicate colonies of variants -2 and -3 changed in appear ance through 15 or more days of incubation, such cultures when young gave useful results as controls on inocula used in various experiments. However, monitoring by use of streak plates is preferable as they indi cate the ratio of variant formation where change has occurred in stocks or inocula. Sectors or outgrowths were probably most common in colonies of Y-23 11-2, less common in Y-2311-3, and not observed in Y-2311-1. Cultures of Y-2311-1 (normal pale pink on ME), -2 (red on ME), and -3 (yellow on ME) were transferred serially twice in 3% glucose Y:YI shaken cultures, the first transfer being 7 da old when the second 349 MYCOLOGIA, VOL. 67, 1975 transfers were inoculated. When the second set of flask cultures were 7 da old, cells from each were streaked on YM and ME plates. 'When the plates were 7 da old, Y-2311-1 had produced completely homo geneous, pale pink colonies on both media, with no formation of variants. Variant Y-2311-3 produced on ME plates only yellow colonies and on YM plates only pink colonies which were a somewhat deeper pink than colonies of Y-2311-1, indicating variant -3 had not changed. Variant Y-2311-2 formed on ME plates about lOro red colonies and 9Oro yellow, but on Y:~\r plates the colonies were about 10% red and 90% pink. Variant Y-2311-2 had converted during two serial YJ[ shaken cultures into about 90% of type -3, Because variant -2 grows slower than -3, a rapid change in types would be expected in serially transferred cultures after the variation had oCC1.1rred. All three variants were tested with elicitors R. glutinis var. glutinis YB-4027 and C. guilliermondii var. guilliermondii YB-+030 on y:yr agar slants. The stronger synergistic reactions were given by Y-23 11-3. Both elicitors gave red color in mi..'<:tures with variants -2 and -3 at 24 hr, YB-4027 producing a much stronger test than YB-+030. It should be emphasized that when 1 da old. Y-2311-2 alone is only pale pink, the brilliant red of this culture when alone requires several days to develop, while ~ynergistically in the presence of YB-4027 the red develops in 24 hr. Perhaps due 'to its slower growth, it gave a weaker test at 24 hr than variant -3. Variant -1 was not synergistically active. Variants Y-2311-1. -2. and -3 were mixed together on YlVr slants in all combinations of two. None showed synergistic action over a period of 7 da.

;\Of)ITION,\L ELICITORS Strains tested on solid Y.l[ 1I1cdilllll.-:\ first selection of 20 strains was made from the genera RllOdotnrllla. CryptocoCCllS. Candida. De baryomyces. Hanse1l1tla. and Saccharomyces. Each strain was mixed with YB-4026 and with Y-2311 red parent strain. and the mixtures were streaked on plates. \Vhen the cultures were obsen'ed at 24 hr. comparative amounts of pigment showed five of the eight nonfermenta dve strains were elicitors. one of the three weakly fermentative strains was an elicitor, and one of the nine fermentative strains was an elicitor. The elicitors are listed in TABLE I in decreasing order of pigment intensity. Strain YB-4027 is included with each screening to show the relative capacities of the strains tested in respect to this most commonly used elicitor. \VICKI::1UiAM AND KURTZ:-'1AN: AUREOBASIDIUM l'L:LLULANS 350

Strain Y-2311 yielded much more synergistic pigment with the 20 strains tested than strain YB-4026 when the mixed cultures were 24 hr old. At this time the mixtures of A. pullulans variants with NRRL YB-3935 and NRRL Y-1400 contained some yellow pigment as well as red; mixtures of the other strains produced apparently only red pigment. The controls, Y-2311 alone and YB-4026 alone, were faint pink with traces of yellow and orange. "Vhen the cultures were 4 da old, YB-4026 remained much inferior to Y-2311 for production of synergistic pigment. A second selection of additional potential elicitors involved 12 :;tr;J.illS of Rhodotoruta, 8 strains of CryptocoCClLS, and 13 strains from a total of 9 other genera (TABLE I). Procedure was the same as before except 28 C was used instead of 25 C and first obsenations were made at 48 instead of 24 hr. As before, synergistic pigment was stronger with Y-2311 than with YB-4026. Other strains were tested subsequently to those in TABLE I. One was NRRL Y-335 Dematium nigrmn (? =d. pullulalls, Cooke, 1959) which seems worthy of note for it produces a viscous, stringy polymer associated with a black pigment, and in combination with Y-23 II pro duced a dark blood red probably due to admixture with the polymer of Y-335. Four normal isolate::; of d. pullulans, NRRL Y-2S6i, YB-J.029. NRRL YB-2552, and NRRL YB-2554, were tested with YB-J.027 and YB-4030 C. gltillicrmolldii var. !]uiilicrmolldii to determine their ability to act as synergists. )Tone did. Strain NRRL YB-40W. an unidenti fied isolate from a beehive, gave strong synergism with Y-2311, weak with YB-J.026. Strain YB-4060 produces black spots on the surface of a sooty-appearing colony that resembles .-1. pll11ulans. It is not a color variant, but it evidently is an elicitor. Over a 2-wk incubation. some mixtures or normal .-1. pl/Ul/tans, as well as some color variants with YB-J.027, on Y:\[ slants slowly turned brown; whereas none of the normal strains :;eparately became brown. This is evidently a favorable effect of the R!lodotorula in inducing the production of chlamydospores and other black or brown cells of the culture and often occurs in mixed flask cultures, as will be noted later. Contaminating mold cultures occasionally have been observed to be elicitors.

Strains tested in liquid media.-Yl\[ and l\IE media were used, and special attention was given to YNB, a chemically defined medium that 351 MYCOLOGIA, VOL. 67, 1975 might facilitate isolation of pigments. Rates of growth of Y-2311-l, -2, and -3 appeared to be comparable in liquid and solid media. heaviest for -1, least for -2, and intermediate for -3. Color synergism occurred

TABLE r SV:-iERGISTIC ABILITIES OF SELECTED STRAI~S OF MICROFU:-IGI TO PRODUCE PtG:'fE:-IT !:-< mXTURES WrTH COLOR VARIANTS Y-2311 RED PARE:-iT A:-iD YB-l,U26 ON Y:VI" AGAR PLATES

Decreasing order oi elicitors NRRL numher

First screening: Rhodotoruia giutinis var. glutinis YB-l,027 Cryptococcus aibidus (Saito) Skinllcr var. aibidus Y-l,*OO Cryptococcus aibidus (Saito) Skinncr var. diffluens (Zach) Phaff et Fell Y-lS17 Candida iipoiytica (Harrison) Diddens et Lodder var. lipoiytica Y-I094 HansenuJa anomaia (Hansen) H. et P. Sydow var. anomaia Y-366 Candida reukauffii (Gruss) Diddens et Lodder Y-1348 RlwdotoruJa patlida Lodder Y-339 RhodotoruJa sp. (faintly pigmented) YB-393S Second screening: RlwdotoruJa rubra (Demme) Lodder Y-1S92, Y-lS94, Y-1S90, Y-lS93 C. aibidus var. diffluens Y-lS0S R. glutinis var. gtutinis YB-t027 Sporobolomyces saimonicolor (Fisher et Brebeck) Kluyver et van Niel Y-8S0 R. gtutinis var. gtutinis Y-I091. Y-lS88, Y-1S96, Y-1S83, and YB-l,363 PrototMca zopfii Kruger YB-990 PrototMca sp. Y-2348 Endom-vces ovetensis Pelaez et Ramirez Y-1S23 Trichosporon capitatum Diddens et Lodder Y-14.87 Cryptococcus laurentii Kufferath et Skinner var. laurentii Y-199 C. aibidus var. diffluens Y-IS0l RlwdotoruJa minuta (Saito) Harrison var. minuta Y-1S89 R. rubra Y-1S9S ToruLopsis candida (Saito) Lodder Y-2-l:S Candida curvata (Diddens et Lodder) Lodder et Kreger-van Rij Y-1S11 Cryptococcus luteolus (Saito) Skinner Y-986 Cryptococcus flallUS (Saito) Phaff et Fell Y-1S8S Trichosporon jermentans Diddens et Lodder Y -1,*92

.. YM - yeast extract-malt extract medium. with YB-4027 and other elicitors in shaken cultures. The colors pro duced in Y1'I and ME liquid media were similar to those produced on the corresponding solid media except for a more brownish trend. When growth in the flasks was allowed to settle, the brownish solids settled to the bottom, revealing more yellow, orange, red, or purple in the WICKERHAM AND KURTZMAN: AUREOBASIDIUM PULLULANS 352 supernatant. Ordinarily descriptions were recorded while the cells were still suspended. Mixtures of Y-2311-2 and -3 with elicitor YB-4027 were highly viscous, especially in ME medium. The mixtures were much more vis cous than the constituent cultures alone. Thus the synergistic color variants also effect a synergistic production of viscosity, presumably through greater production of e.xtracellular polysaccharides. YNB medium with 2, 3, 4, 5, and 8% glucose was studied for pigment production using YB-4026 and elicitor YB-4027. Incubation was at 28 C on a Gump shaker. At 48 hr the 2% glucose culture of YB-4026 + YB-4027 had the most orange-red pigment. the 3% glucose culture had the ne.xt most, and so on, the pigment decreasing with in creasing sugar. At 72 hr the 2% glucose culture had turned brownish, and the 3% less so. The 4% glucose culture had gone beyond the brilliant orange-red state, the 5% glucose culture had reached its maximum pigmentation, and the 8% glucose culture had not yet reached its prime color. Thus the 2 and 3% glucose gave optimum pigmentation at 48 hr; the 4, 5, and 8% glucose presumably gave more pigment but required increasingly longer incubation. Occasionally, additional strains were tested for their ability to elicit increased pigment by synergistic color variants of A. pttllttlans· in shaken media currently in use at the time. Some were selected because they had given interesting results on solid media; others for possible potential in industrial use. Nearly all were nonfermentative. . Candida lipolytica var. lipolytica NRRL Y-1094 was tested in 2% glucose medium on a Gump shaker at 28 C. It was mixed with Y-2311 red parent culture and at 3 da the mixed culture was tomato-juice red. A comparable control mixture was Y-2311 + YB-4027 which became reddish brown. After settling, Y-2311 + YB-4027 had con siderably more soluble pigment in the supernatant than the mixture containing Y-I094. A control culture of YB-4027 alone was orange but had no soluble pigment. Other food and feed yeasts found to act as elicitors are Candida utiZis NRRL Y-900 and Saccharomycopsis fibuligera NRRL Y-I062. Saccharomyces kluyveri Phaff, Miller, et Shifrine NRRL Y-2488 unisexual diploid 7DI, a fermentative yeast, was mixed with Y-2311 red parent and shaken for 5 da on a reciprocal shaker at 2S C. The media used were 3% glucose and 3% sucrose in YM. They yielded about equal intensities of a maroon-ehocolate brown color. The two 353 MYCOLOGIA, VOL. 67, 1975

flasks with Y-2311 alone had only a light pink-orange color. Strain Y-2488 is not pigmented. Some colorless algae and a green alga were tested for their ability to act as elicitors in 3% glucose Y:\I medium shaken for 5 da on a reciprocal shaker at 25 C. The colorless algae were YB-833 Proto theca mon'f01"mis Kriiger, YB-990 P. ::opfii. YB-4330 Prototlzeca wickerhamii Tubaki et Soneda, YB-4825 Prototheca ciferrii Negroni et Blaisten, and YB-4826 P. .:opfii. Ta.,,

Strain NRRL Y-1592 R. rubra was one of several strains of Rhodotort£La that gave stronger synergistic reactions than YB-4027 on YM agar. Strain Y-1592 was compared with YB-4027 as elicitors for Y-2311 red parent and YB-4026 A. pulluLans. Growth was in 2% glucose YM medium on a reciprocal shaker at 28 C. At 48 hr'the culture of Y-1592 alone was reddish but vastly inferior in color to the mixture~. The supernatant of culture Y-1592 + Y-2311 W:lS dark red and the abundant sediment was a dark brown-maroon. Culture Y-1592 + YB-4026 also had brown-maroon sediment but less deeply colored. At 72 hr both supernatant and sediment were brown. but the sediment of Y-1592 + Y-2311 was black, and the supernat:lnt was dark brown. Control cultures YB-4027 + YB-4026 and YB-4027 + Y-2311 at 48 hr were both red. but YB-4027 + YB-4026 had a more lustrous or fluorescent appearance, possibly due to the presence of more yellow pig ment. Both of these rultures had a thin, transparent. deeply pigmented ring about 30 mm above the surface of the culture when at rest: after 24 more hours of shaking. these rings were bright red. tnnsparent translucent. with the appe:lrance of having st:lrted to run clown the w:lll of the flasks. but they were hard and resistant as though they were a natural transparent plastic.

E fleets of temperature on pigment· formation.-:"fixtures of Y-2311 with YB-4027 on Y:Yl agar were incubated at 5. 15. 25. 28. and 35 C. Observation extended over 10 da and the g-reatest development of color occurred at 25 C, followed by 15 and 28 C. and then 5 C. There was no color development at 3S C. and growth at 5 and 3S C was about half that at 25 C. When Y-2311 and Y-2311-2 were incubated nn Y.\f a!:;:1.r at 35 C for 1. 2. or 4 da and then removed to 25 C. the red color cle\'eloped within 24 hr while control cultures at 25 C remained p:lle pink. Incuba tion for 2 cia at 3S C gave the greatest colnr enh:lncement. Within 24 48 hr after the red developed. it began to fade. and the cultures became a progressively fainter yellow-orange that never entirely disappeared. Strains YB-4026 and Y-6754a showd this phenomenon to a lesser de!:;ree than Y-2311 and Y-2311-2. ;,-fixtures of these cultures with YB-4027 showed no color enhancement at 3S C. but the typical color formation occurred when they were removed to 25 C. The pigment formed by the strains first incubated at 3S C and then transferred to 25 C was :1.bn\1t half as intense as when the elicitor strain was present. 355 MYCOLOGlA, VOL. 67, 1975

RUDIMENTARY CHEMICAL OBSERVATIONS Change of color with change of pH.-Addition of acid to culture:; of the variant color strains and their elicitors produced no change, but addition of base caused a loss of both red and purple. The colors were imme diately restored by neutralizing the base with acid, and the process could be repeated several times with the same culture. The results with color variants Y-2311, Y-6754a, and YB-4026 along with elicitor strains R. glz£tinis var. glt£tinis YB-4027 and C. luteolus NRRL Y-7471 are given in TABLE II. Marked, immediate increases in viscosity accompanied the addition of 1 N sulfuric acid to liquid cultures of variants Y-2311-2 and Y-2311-3 when tested for change of color with change in pH. Increase in viscosity also occurred when sulfuric acid was added to a 4-da-old shaken culture of Y-2311-3 + YB-4027.

Iron as an elicitor.-Small pieces of metallic iron added to YM agar cultures of Y-2311 caused growth near the iron to become a deep tannish purple. whereas metallic zinc had no effect on color development. Con centrations of 10-2 to lO-s ~r iron and zinc as ferrous ammonium sulfate., Fe(NH4 hCS04h'6HzO, ferric ammonium sulfate. FeNH4 (S04h' l2H20, and zinc sulfate. ZnS04'7HzO. were added to YM agar and the pH was adjusted to 6.5. Plates containing these media were inoculated with Y-2311 and a mixture of Y-2311 and YB-4027 and incubated at 25 C. The results were the same on the ferrous and femc iron plates where a color change was noted after 24 hr. At 10- 2 M Fe. the growth of both Y-2311 and the mixture with YB-4027 was a deep purple-brown and became progressively lighter as the iron concentration decreased until growth on the lO-s M Fe plate was no different from the YM con trol plate. While the color induced by iron was as intense as the color of the mixture on control plates, it was not red like the control mixtures, nor did the red develop in mi.."'tures on the plates containing iron. Zinc had no effect on color development, although 10-2 M Zn was markedly inhibitory to growth.

Oxidation and reduction.-Two parallel streaks about 15 mm apart, one of cells of Y-2311 and the other YB-4027, were made on a YM plate. Before the cultures had made contact Y-2311 was deep red. especially on the side toward the Rhodotontla. Three percent hydrogen peroxide was placed on Y-231 1. Part of the growth became white, but farther away from the point of application the growth was light red. A ~ n... ~ TABLE II > ~ COLOR CIIANGES I'UODUCED DY ADmTlON 01' ACID AND BASE TO 4-DA-0I.n YI\! AGAIl-GIWWN CULTUllES OF SYNERGISTIC I'IGME~T-I'ROJ)UCING S1'RAINS AU)NE A~O WITU E1.ICITOIlS 1~1I01l0TOIlUL.A GLUTINIS > VAR. GLUTll"IS Y8·4027 AND CRYl'lOCUCCUS I_UTEOLUS Y-7-171 §

Cultural appearance aher u,lditlon of acid or hase ~

------.- c: NRRL numbers Cutural appearance ~ Addition of Addition of j N lIel to cnltures Addition of J N hOIl N j N IICI /irst treated with j N KOII k" >R Y-2311 Palc pillkish whilc No challgc Palc yellow Oril(inal color retnfllcd 2: Y-2311 + YB-4027 Inlcnsc dark rcd No changc Browllish Iliad: Oril(inal color rctllfllcd Y-HII + Y-7-l71 Dark red No challgc Brownish Illack Oril(illal color rctllfllcd > YB-·J(l26 I'alc pink No challgc Palc }'cllow Oril{inal color retllfllcd c: Y11--1026 + Y B··I027 Dark red No challgc Browllish hlack Origillal color retllrlled ~ YB-4026 Y-1471 Dark red No challgc Browllish black Oril(inal color relnflled o + b:l Y-675b 1_avelltlcr No changc Pale brownish pllrple Oril(inal color rclllrllcd ) Y-675-la + YB-·I027 1lark pllrplc No chanl(c Brownish pilI' pic Original color rclllfllcd Ul Y-675-la + Y-1471 I'lirple No challgc Brownish pllrple Oril(illal color n:tllflu·t1 ...a VB--11m Orallge No challge No challge No ehallgc c: Y-7-171 I.ighl lallllish rcllow No challgc No change No changc ~

~------~-_.. _------.-.------_.. _---- '0 c: r' r' c: r' )- 2: UJ

0.1 UI Ch 35i MYCOLOGIA, VOL. 67, 1975 few small crystals of sodium hydrosulfite, Na2S 204' 2H20, were placed on the white and semibleached areas. The latter became deep red again in a circle around the crystals, but the white area did not become colored. Slant cultures of YB-t027 and Y-2311 , separately and mixed, are catalase positive. The evolution of oxygen from 3ro hydrogen peroxide was especially strong with Y-231 1. The red pigment was decolorized.

DISCUSSION Strain YB-4026 was the first synergistic color variant isolated at the Northern Regional Research Laboratory. It becomes yellow on both :vIE and Y?vI agar. The color soon turns to light pink on Y:vI medium but retains its yellowness on :vIE, possibly due to the greater acidity of this medium. The yellow type tends to shift to an unstable red type that is red on both ?vIE and Y:Y! media. Both yellow and red variants produce much more red pigment on YM agar when grown in mixed cultures with many other microfungi. A red variant type Y-2311 was isolated from frass of a coniferous tree. From it were obtained a yellow variant Y-2311-3. a red variant Y-2311-2, and a normal A. pul/ulans, Y-2311-1. The color variants of Y-2311 were more chromogenic than YB-4026, and the red type, though frequently changing to the yellow type. was sufficiently smble to permit work with it. It grew more slowly and was more strongly filamentous than were the yellow type and normal A. pttllula1ls. Strain Y-6754a is a synergistic color variant that produces a purple pigment. In general. the color variants are similar to other strains of A. pttllttlans in morphol ogy and assimilation of carbon compounds. Solid ME medium alone is adequate for distinguishing between variants Y-2311-1, -2, and -3. and the synergistic color variants -2 and -3 are named red and yellow according to their respective colors on this medium. Additional media show other color aspects or phases of -2 and -3. As examples, ME is more constant in maintaining the yellow .of Y-2311-3, but this variant on Y:\J agar soon changes from yellow to pink. The red variant is red on both :vIE and Y:\'I, and shows little evidence of a yellow phase on YM, but a similar medium, e."<:cept that it contains 2% sucrose instead of 1% glucose and lacks malt extract, brings out a yellow stage clearly which later changes to red. Many colors and sections observed in triplicate colonies of the color variants when 15 da old may be due largely to changes in pH and to mutations. Initial variations in wild type toward the more chromogenic smte shown by maintainable color variants may possibly be of slight degree and easily lost. Strains that produced weak to strong yellows and reds WICKERHAM AND KURTZMAN: AUREOBASIDIUM PULLULANS 358 have been isolated and lyophilized, yet when cultured from the lyophi lized state produced neither a yellow nor a red growth. Strain YB-4588 was originally such a red culture; strains YB-4587 and YB-2554 were originally pale yellow cultures. However, considering only strains YB-l-026, Y-231l-2 and -3, our impression is of a relatively rapid shift from one strongly pigmented color form to the other, accompanied by minor changes in morphology and growth rate, whereas reversals to the wild type were confirmed only once, in a culture of Y-2311 red parent maintained under oil at 8 C. The unstable red phase of YB-+026 was observed to be produced hun dreds of times in a single culture, but a relatively stable red form. com parable to Y-2311-2, was being approached but had not been obtained when further work to stablize it was discontinued. .AlI the synergistic variants of A. p1tllulans studied were obtained from samples collected in subtropical or tropical areas. The species grows poorly or not at all at 37 C. Continued e."<:posure to high tem peratures, accentuated perhaps by toxic habitats such as pine pitch and its terpenoid constituents, may induce the formation of synergistic color variants. vVe believe the induction of variants may be more successful using cultures isolated from natural materials recently acquired in tropi calor subtropical countries rather than using old laboratory cultures. Several factors complicate analysis of synergistically produced colors in cultures. Among them are the following:

1. The unknown number of chemically distinct pigments that each variant has the ability to produce. variations in media that cause or inhibit their production. and the variations in color that may occur with varying ratios of pigments. 2. The rate at which one color variant changes to another color vari ant and the additive effects of the colors they produce. 3. The ability of the pigments to change color with changes in /' H. or to become decolor1zed by changes in pH. or tn he affected hy oxida tion-reduction potentials or by other types of chemical changes in the chemically reactive color molecules. 4. The ability of color variants to produce colorless compounds that may become colored by changes in pH or other chemical changes. 5. The influence of black and brown produced by aging of cultures of A. pullulans color variants, especially as they are influenced by Rhodotorula strains used as elicitors and bv the medium. 6. The obstructions to pH changes or pi~nents that are inside living cells, especially those having capsules. 359 MYCOLOGIA, VOL. 67, 1975

7. The influence of pH and chemicals on solubility of pigments. 8. The influence of color produced by pigmented elicitor organisms.

Although some factors influencing colors of cultures have been e."<:amineu in a rudimentary manner, no study of the color chemistry was attempted. The influence of temperature on color clevelopment may offer a clue to discovering the pathways involved in pigment forma tion. When cultures were incubated at 3S C, there was no pigment forma tion even in the presence of elicitor strains, but upon removal to 25 C color variants became pigmented when alone as well as when with an elicitor although the variant strains grown alone soon lost much of their pigmentation. Variant strains grown near an elicitor became pigmented, especially on the side facing the elicitor. This unequal pigmentation sug gests diffusion of a pigment precursor from the elicitor and further that this precursor cannot be metabolized to form pigment at elevated tem peratures. Since color develops upon removal of cultures to lower temperatures, the color variants themselves must be able to form the pigment precursor and then this precursor accumulating at elevated temperatures becomes available for pigment formation at lower tempera tures. Disappearance of the pigment after a few days may indicate that the pigment is being degraded for the precursor, which has an essential metabolic function elsewhere. While much of this is conjecture, other observations tend to support this hypothesis. When color variants are stren~ed on plates and indi vidual colonies are crowded, they are usually more highly pigmented than when widely spaced. Thus not so much of the proposed precursor is needed for growth and betomes diverted to pigment formation. The highly pigmented sectors which sometimes develop in colonies of color variants frequently grow at the same rate as the rest of the colony and may represent cells that become genetically den:pressed for greater precursor formation or that have acquired this characteristic from either a favorable heterocaryotic recombination or from the inclusion or re moval of an e."ctranuclear genetic factor. Color variants and elicitors may be selected for the production of a wide array of colors including yellow, orange, red, purple, brown, or black. The colors could be varied by changes made in pH of the cul tures during or at the end of propagation. To varying degrees the pig ments are also susceptible to color changes by oxidation and reduction. Yeasts studied synergistically form e."<:tracellular polymers that increase viscosity of the cultures, and viscosity is increased further by adding VVICKERHAM AND KURTZMAN: AUREOBASIDIUM PULLULANS 360 small amounts of sulfuric acid to mature cultures. The aforementioned capabilities would suggest that feeds, foods, and food colors finding their greatest use in Oriental countries could be produced commercially by careful selection of well-balanced pairs of color variants and elicitors. Candida utiZis and Saccharomycopsis fibuligera, both involved in food production in Sweden and the Orient, are elicitors. The best elicitors have been found among species of the genera Rhodotorula and Cryptococcus. Lacking the ability to produce a gaseous fermentation, they conserve available carbohydrates for growth and pig ment formation that fermentative yeasts would partially convert to car bon dioxide. Rhodotorula additionally produces carotenoids that in crease both the nutritional quality of the food and its color. Some strongly fermentative yeasts, such as 5. kluyveri, are also elicitors. Cultures of color variants were observed to contain e.'<:ceptionally large e.'<:tracellular bodies composed mostly of lipids but containing globules and granules. These bodies are believed to be comparable to the lipid droplets observed by Ruinen and Deinema (1964) in cultures of R. glutinis, A. puiZztlans, Candida, and Cryptococcus. The extrace!!ular droplets they observed occasionally agglomerated and formed larger droplets. some even visible to the naked eye. A green alga, strain YB-3399, that grows wel! on organic media and also lyophilizes well, strongly stimulated the production of pig ments by the color variants of A. pullltians. .-\s it grows more slowly than most yeasts. it might be desirable for large-scale production to grow this elicitor or other slow-growing ones separately for se\'eral additional hours before adding to the culture of the color variant.

LITERATURE CITED

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