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<ed on YM plates in an attempt to obtain pure cultures of the orange variant. The streaked plates produced many colonies that were not orange but pink, and two colonies had tiny red spots that appeared to be secondary growths at the centers of the colonies. Attempts were continued with considerable success; yields were obtained of about 80% colonies having blood-red centers and 20% dark and lighter red colonies lacking secondary growths. No further attempts were made to obtain a pure red form of YB-4026 after the study of red strain NRRL Y-2311 was commenced, because it produced more stable 345 :Y{VCOLOGIA. VOL. 6i, 19i5 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.