2 8 2 5 I~ Illp·8 2 5 1.0 :~ 11111 . 11111 . 1.0 I~ 1= 11111 . ~ II~-~ 2.2 I~ IIli?~ 2.2 Ui I" ~ I~ I; I~ w J:Z ~ 1,1,g ~ m,t"g 2.0 LiI. '" ... " 1.1 ...... "" 1.1 ...u,~u ------

11111 1.25 1111,1.4 111111.6 111111.25 !lII,1.4 111111.6

MICROCOPY RESOLUTION TEST CHART MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDS-J963-A NATIONAL BUREAU OF STANDARDS,1963-A ~AROTENOIDS

in the FUNGI e­ :..~

CJ")coMUCORALES .~, ~ .',:.; ,

"

Special Reference to Choanephoraceae

U.S. DEPARTMENT OF AGRICULTURE AGRICULTURAL RESEARCH SERVICE CONTENTS Pllg" INTRODUCTION ______

CAROTENE IN ]\I[UCORALES______• ___ ••• ____ 2

FUNCTION OF CAROTENE IN SEXUALI'l'Y OF l\l{ucorur,J.JS______7

CAUOTENE IN THE FAMILY CHOANEPHOHACEAE______9 OeeulTcnce and History of the Chonncphora(,Nl(' ______I0 Life Cycle_---- ______., ______14 Cnrotene in Chonnephorneelle______17

LITEHATURE CITED______23 Cllrotene in l\{ucornil',s______.______2:~ Oc('ulTcnce llnd ]~ift· l 'yell'S of C'hoanephomeell(' ______ao

This review wus madc ns pIU'L of the irl\resLiglltions bl'ing eonducted at the Northern Hegionnl ]{csellJ'eh Laboratory on indust.rial uliliZtl­ tion of CCI'CIlI gmins. The ARS Oultmc Collection, mnintlline(\ there, is one of thc world's IUl'gest and most ('omplctc ('ollections of industrially important, uILCtcl'in" molds, Ildinornyectes, n,nd yCilStS. The Collection serves as n, sourcc of ItlIt,hcntic miCl'o-oJ'gnlli~ms for the fermentative production of orgillli<.~ neids, vitnmins, Illltibiotics, enzymes, feeds, bcvemgcs, Ilm\ foods.

Washington, D,O. Tssu('d August 1\)(11

For RIlIl' by the Superintendent of Doculllents, U.S. Government Printing Oflicl' Washington 25, D.C. - Pric(' 15 cents II •••• C,AROTENOIDS ht the FUNGI MUC,ORALES Special Reference to £hoanephoraceae fly C. W. HESSELTINE. Read, ARS Culture Collection, Fermcntation Laboratory, Northern Utilization Research and De\'elopment Division, Agricultural R(,­ scarch Service 1

I~TRODUCTION

Mycological aspects of the fungi Mucorales and Choanephoraceae are emphasized in this review. 'Most of the chemical aspects of carotene and the theories of the metabolic pathways in its for­ mllqon were intentionally omitted. Books Itbout the chemistry of cnrotenoids have been written by Goodwin (44) 2 and by KalTer and .Tucker (79). Their terminology is used throughout this re\'ie\\". One charactp.ristic of many of the Mucorales is the yelIo\\' or orange pigments within the fungus mycelium, espeeiltlly the substrate myce­ lium. Aside from these two colors, pigments in the order nre restrict­ ed to blackish ones with but few exc-eptions. In a few spccies the yellow color hns been identified ns carotenoid in 11Ilture, of ,,-hich the mnjor proportion is ,B-cllrotene. This mllterinl, in addition to its ap­ parent importance to the organisms, espeeinlly in sexual reproduction, has certuin practical implications. Isler (78), Bunnell (21), !lI1dBIlU­ ernfeind (8) and their coworkers touch on the use of carotenoids in foods. Colors are desired in some foods for better eonsulller lH'('l'ptllbili ty. Since the fat-soluble cllrotenoids are yellow, orange, flnd red, they arC' excellent for this purpose. One method of inC'orporating the ,B-cltrotenc in fat-base foods is to micropulverize it lLnd to suspend itin edible faL. In this form it could be used in stich foods as margarine, bull('r, shol'tC'ning, pl'oeC'ss eheese, egg yolk pl'ociuels (both frozen and driC'(i), and bakery prod­ ucts. It migh t be used in other' Joods wiJrrc color is c\rsimble sueh as in macaroni, sClllolilHt, breaded food products like fish sli<'ks, li'rC'lwh dressings, whipped erellll1 and loPpin6"S, and pOpCOrIl oil. Recently BUllnell Illld others (21) showed tlllll ,B-<'nrot('nc may be prepltrcd ill

I The Northern Regional Rc-sC-llreh Laboratory at Pc-orin, m" is headqllllrtf'r, for the Northern Utilization Hescarch and De\'clopmcnl Dh·ision. 2 Italic 11l1mbc-rs in pilrcnthcses refer to Litemllll'(' Cited, p. 23. 1 2 TECHNICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE a stable, dry, bea(Uet form for coloring water-base foods. In this form it could be used in such foods as citrus beverages, primary cheese, egg yolks (frozen and dried), eggnog, ice cream, dry cake mix, puddings, and gelatin desserts. A second method of adding pigmentation to foods would be to in­ troduce added carotenoids in a crude form into a:Jimal feeds to give more color to poultry, eggs, and beef. Another important considera­ tion is the fact that ,8-carotene when ingested by many animals, in­ cluding man, is converted into vitamin A. At present two sources of carotenoids are available-natural and synthetic. They occur in lUltural materinls, such as ,8-carotene in dairy products, lycopene in tomatoes, and zeaxanthin in yellow corn. Commercially, large amounts of alfalfa are used as a source of natuml carotenoids. BeUl-carotene is being produced in large quantities by chemical synthesis. It might be possible to utilize a third source-fun­ gi-especilllIy of the order 1lucorales, either as a crude fermentation product for feeds or as a pure compound by extraction of tbe ,8-caro­ tene from the mycelium. CAROTENE IN MUCORALES

Tn 1890, Zop[ (111, p. 148j stat(,cl thilL It ycllow [itt material, prob­ ably lipochrome, was pl"Csent in tho sporangiophores, spomngia, and spores of Piloboius and n/ucor. He stat('d that none of it had bc£'n isolat('d and its exact nature was not kno\\n. Two years later, Zop[ U12) studicd the ye11o\\- and ornngc-red mil terials of the ~[ucorules in Pilobol1lS, especially P. kleinii. He illustrated the distribution of pigments in the myc('lium and describ('d tlwir solvent extraction. By sp('ctrogmphic means he showed also that ahsorption bands occurred at 484-469 imd 452-430. By this and ciwmical means he concluded that tho pif!:ll1onts wero Cn1'otelH'S. The same materials \\'01'0 found in P. oediZJUs nnd P. cryslallinus. The carotenes were pr('sent in the spomngiospores and were also associated with the f!ltly Il1fltorilll in the zygospor('s. In Zopf's opinion the carotenes wen~ r(,<;('1"Y(' foods since they aml the fatty material disn.ppeared wi th til(' germination of spOTes. Kohl (81) accepted the SltmO icleft, althouf!:h othors, sllcb as Pitlmer (02), believed thn,t more likely the cnl"Otenoids were only associated with food 1'PSefVes. Yiln ,Vissolingh (110) showed that carotone \nlS present in Jlucorjlnnl8. A stud:y of carotpl1(,s in ~rllcomlus has recontly been undeetaken primarily in the s()('cies Jf1lcor hiemalis flnd Phycomyces blake8leean1l.~ and eyen more rec(1ntly at our labomtory on members of tbr Cboll­ nephomceao. En,llior rovio\\-5 on Lhe occurrenco of Cltrotnnc in Mucomlcs arc by Schopfer (103) and Goodwin (,48, 44). CAROTENOIDS IN THE FUNGI MUCOHALES 3

Chodat and Schopfer (29) proved that the yellow pigment in J.\!ucor hiemalis was a carotene. Schopfer (102) showed that is-carotene was present in Phycomyces blakesleeamlS and determined that either asparagine or glycine could be used as a source of nitrogen for growing the fungus for producing carotene. Schopfer and Jung (108) further found that the is-carotene in this organism had vitamin A activity in feeding e:\.-periments. Castle (25) also reported the presence of a-carotene in Phycomyces. Bunning (18-20) reported carotene in the sporungiophores of Pilobolus kleinii. In a study of Phycomyces, KalTer and Kruuse-Voith (80) reported both is-carotene and a-cm·otene present in the mycelium. Bernhard and Albrecht (.9) studied lipids of Phycornyces blakesleeamlS and reported pigments-possibly is­ carotene, a-carotene, ano lycopene. Schopfer and Grob (104) n,nd Grob, Schopfer, and Poretti (69, 70), studying Phycomyces blakesleeaml8 and J..l1ucor hiemaliR, fOlllld that a medium having lactllte as a sole source of carbon permitted only slight growth and no carotene formntion. vVhen acetate was added, growth and carotene formation \\'cre stimulated. Later, Schopfer and Grob (105) demonstmtrd that a medium composed of sodium acetate and ammonium nitrate supportcd growth of this fungus and allowed nbundant formation of Cllrotenc. 'Yhen acetate was the solI' source of carbon, it acted as It preclIrsor of carotene. This finding was con­ firmed by Friend, Goodwill, and Griffiths (36,37), who stated thnt lilly member of the tricarboxylic acid cycle added to It medium containing glucose find ammonium nitm,te stimulated growth equally ,,-ell, but that varying degrees of is-carotene production were due to vllria­ tions in the finnl pH values of the medium. The addition of .B-ionone stimulllted carotenogenesis from 200 to 300 percent in an Ilcetate mcdium. The addition of .B-ionone had earlier been shown by '\[11C­ kinney and others (87) to stimulnte .B-Citrotene production. The Swiss workers (68, 71) had siJo\\-n that labeled CJ.! was present in CH3 lind coon groups and that these carr-n ntoms were incorpomted equ,llly into the .B-camtenc molecule. Further information on the carbon from acetllte is given by Grob and BUtler (58, 59). Reichel and Wallis (95) have shown that P. blake81eeaml8 formed .B-carotene fmm a vllL"i­ ety of t:cids, including among others citric, succinic, and pyn1vic, and earlier (9.1,.) discllssed the metabolic pathway in the formation of .B-carotene in P. blakesleeaml8. In 1951, Good\\"in and his associates initiated a series of studies on carotenogenesis using P. blake8leeanu8. The selection of this organ­ ism to study carotenogenesis was bnsed on the following reasons: (1) The fungus is non photosynthetic. (2) It is ensily cultivated on a simple aqueolls medium with vitnmin BI as the only miemll1ltrient required. (3) It produces rather large amounts of .B-carotene. 4 TECHNICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE

In the first detailed work, Garton and others (38, 39) showed that in this organism {3-cltl'otene was the prcdominant pigment although a-carotene also occurred. No xanthophylls or lycopene were found. Schopfer's medium was used; it contained: glucose, 10 percent; L-as­ paragine, 0.2 percent; KH2P04, 0.15 percent; lvIgS04 ·7H20, 0.05 per­ cent; and thiamin, 25 /lg. Schopfer's medium gave maximum growth, and lipid production occurred in 5 to 6 dl1Ys, but maximum carotene production did not occur until 7 to 9 days after· inoculation. The carotene content began to diminish by the 14th to 16th day. When grown in the darki the fungus contained only about half as much {3­ carotene as when grown in the light. These investigators also found that the - strain produced twice as much (3-carotene as did the + strain. Goodwin and Lijinsky (50) showed that the L-valine or L-Ieucine, when used alone or with glycine or L-asparagine, stimulated caroten­ ogenesis sometimes as much ns four times. The minor polyene components of P. blakesleeanus were also studied by Goodwin (,41,42). Besides a- and {3-carotene the following materials were found: phyto­ fluene, 2.0 percent; 'Y-carotene, 0.85 percent; .I-carotene, 0.35 percent; neurosporenc, 0.6 percen t; and lycopene, 0.6 percen t. The percen t­ ages r('presen t the totnl polyenes presen t. Seveml other materials were pr('s(,l1 t that were not posi tively iden tified. 'When diphenylamine (l/40,000) was added to cultures, it nllllost completely inhibited production of the most unsaturat('d car·ot('nes (a-, (3-, 'Y-caroLenes and lycopene) while stimulating production of the more snturated com­ penen ts, phytofluene, .I-carotene, neurosporene, and possibly phytoene. The nitrogen met!lbolism of P. blakesleeanw3 was further studied by Goodwin and Willmer (53) ancl Goodwin, Lijinsky, and 'Yillmer (51), who showed that carotene was made only after the myccliill mat was fully formed. Furthermore, well-formed mats of mycelium dis­ similating glucose could synthesize reillti\rcly more carotene in the absence of llssirnilflble nitrogen than in its presence. The efI'ects of possible carotene precursors on growth, lipogerl('sis, and carotC'nogenesis were studied by Goodwin, Lijinsky, and ""'illlllor (52) and by Glover, Goodwin, and Lijinsky (40), but no substancc tested g!LVe enhanced carotene production. Friend and Goodwin (35) showed that in P. bfakesleeanu8 temperature did not affect the types of carotene produced ill the mnge from 5° to 30° C. The optimum temperature fOl' carotene production was 25° C. Goodwin (42) showed that diphenylamine inhibited the synthesis of the IetlSt saLurlltC'd polyenes in P. blakesleeanus with an increased syn thesis of the most sitturated polyelles, such as phytofluene. Additional study of this aspcct was rcported by Goodwin and othel's ~1-7) and Goodwin, Jllmikorn, ilnd Willmer (49), who showed t.hat in CAROTENOIDS IN THE FUNGI MUCORALES 5 this mold the saturated polyenes were produced much more rapidly than iJ-carotene. Thus, such materials as phytofluene reached their maximum in 3 to 4 days while ,B-carotene reached its maximum in 5 to 7 days of incubation. The increase of phytofluene and the decrease in ,B-carotene were directly proportional to amounts of diphenylamine added. When mycelial pads of the mold were transferred to non­ diphenylamine medium con taining large amounts of phytofluene, thflY did not convert phytofluene into ,B-carotene. Recently, Varma and others (109) studied the effect of starvation iI,nd of diphenylamine addition on the production of various caroten­ oids in P. blakesleeanus. In 1952 the Oalifornia group (87) began studying ,B-carotene in the same organism. They demonstrated that the addition of f:-ionone to cultures of P. bla,kesleeanus at the rate of 2 ,Ltl per 20 ml. of medium, added at 72 hours, incr'3asfld the ,B-caro­ tene from 91.2 to 218 ,Ltg per gram of dry mycelium. However, add­ ing ,B-ionone resulted in depressed culture development.

Later work hy ~Iackinney ilne! OthOl'S (84) showed that met.hyl­ heptenone brought about a reduction in ,B-carotene formation in this organism, a markC'd increase in phytofluene, and the appefimncc of s-carotene. Mackinney and others (88) postulated that the ,B-ionone, or a large portion of it, is incorporat('d into the carotene molecule. According to Chichester and others (26), wben ,B-ionone was added to P. blakesleeamls culturC's grown in the dark, yields of 678 ,Ltg per gram were obtained while those in light produced 869 ,Ltg per gmI11. How­ ever, ,B-ionone was more effC'ctivt) in stimulating carotene formation thnll was light. Furthermore, these authors demonstmt('d that wh('n lnbC']ed sugnT 'I-US incorpomt('d into the medium, the organism prcf­ erentinJly us('d nonsugar cilrhon for the carotene molecule. Chichester and otbers (27) showed that botb aspamgine- andleucine-contnining culture media with ,B-ionone gave high yields of ,B-carotene. Using In,beled glycine, ~hckinney nnd others (86) stated that there appeared to be incol'poriLtion of ltd)('lC'll 0 in to ,B-cm'ot('ne from glycine. Mix­ tures of ,B-ionone and Itwthylbeptenone act independently on caroten­ oid production in P. blakeslee anus according to ).lackinney llnd others (85). Nakayama nnd others (91) prov('d that rnethylheptenonc ncted on P. blakesleennus fiS a powC'rful stimulcnt for phytoene production. Thus the aSPilragine control medium gaye 314,Ltg of phytoene while the 6 'fECRXICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE asparagine medium and methylheptenone gave a yield of 2180 p.g. Chichpster and others (28) reported that carotene produced on a glu­ coso-glycine mC'Cliurn had 38 of its C atoms derivpd from glucose and the wmaining 2 from the methyl carbon of glycine. Grob, Bein, and Schopfer (57) found that P. blakesleeamls, when grown on a medium contt1ining 0.2 percent ammonium lactate to 1 percent sodium acetate as sole Nand. C sources, ga\re the usual amounts of fat, sterols, and carotenoid pigments. Grob and Yon Beust (71) haye shown that three nOIl·.,u·otenoid pigments are present in Ablcor hiemali~. Grob and others (67) showed that pantothenic acid, pantothine, and phosphorylated pantothine increased caroteno­ gellPsis in j\1. hiemalis. The role of pyruvic acid in the biosynthesis of carotene was studied by Grob and others (66). Grob, and Grob and Butler haye published a series of papers on the metabolic path­ way of carotene formation in 1~I. hiemalis (54, 55, 59-66). The effect of antibiotics on carotene formation ill Phycomyces blake8Zm17l1lB has been reported by Goodwin and Griffiths (45). Strpptomycin inhibited cnrotpflogenesis when the organism was grown under n01'mnl conditions in light. The reduction in carotene with any amount of streptomycin was never more than 60 percent. The same y('a1' Schopfer !tnd others (106) confirmed this work. Chloro­ mycetin also inhibited carot('nogen('sis, but t('tronic acid nnd penicil­ lin did not inhibit earotenogen('sis in P. blakesleea.mLS (107). Brucker (17) compnr('(1 phenol and carotene formation in P. blakesleeanu8. Lat('r, Goocl\,-in, Griffiths, and ~fodi (48) report('(1 that high con­ centrations of str('ptomycin did not inhibit growth of P. blakesleeall1l8 in n glucose-aspal'llgine medium but thnt it did inhibit cal'Otenog-en­ ('sis. 1Yhen aspttmgine WfiS 1'('plnced by Iln ammonium salt, inhibi­ tion did not occur. ,\Yhen ,8-ionone was used to stimulate cnrot('ne product:on. the addition of stl'('ptomycin also inhibit('(1 carotene prodlwtion. Thrse illithors also found that chiornlllph('nicol, p('ni­ cillic acid, and terrestric Ilcid inhibited c!trotenogen('sis to 11 grellLer extent than they inhibited growth. Recently, using "A1ucor hiemalis, Grob (56) has shown that men\,­ Ionic acid at ley('ls of 0.5, 1.0, find 2.0 mg per 25 cc. of nutri('lIt solution increased carotene formation. Furthermore, mc\-alollic acid was incorpomted into the ,8-carotene molecule. About the Sllme time Brnithwaite nne! Goodwin (1/J) showed thnt meyalollic acid (,8:0­ dihydro:,:y-,8-methyh·/tleric acid) was as effici('nt 11S leucine in diluting ou t labeled ncetate incorpomted in ,8-carotenc from Phycomycfs blakesleemws. Lsing the samc organism and caI'l'ot slices, Lh('se authors (16) have shown that mevalollic Hcid was incorpornted in the ,B-cttrot('ne 11101('cule. Ritter (.97) reported the pr('s('nce of 11 yellow pigment in "A1ucor adl:entitiu.s wbich he believed W!iS a carotenoid, CAROTENOIDS IN THE FUNGI :MUCORALES 7

Monch (89) proved that this pigment in 1.11. adventiti1lS WfiS indeed a carotenoid. Reichel and Witllis (96) reported that, when + i1nd - strains of Phycomyces were mated under the conditions used by Barnett, Lilly, and Krn,use (7), no increasc in ,,-carotene was encountered. So ffir as is known, no onc has !lttempted to increaso carotene in !lny of tho .Mucomles by menns of inducod mulltlions. BlfLkcslee (12) obtained both stnble and unstable natural mutants of Jo.lucor gene­ vensis, and he reported that BUl'geff obtllined mutants of Phycomyces but cited no rr£o1'ence. Bonner ilnd others (14) showed with ultm­ violet-induced mutants that tho amounts of {1-carotene produc(1d could be markedly Illt(1red in another group of micro-organisms. Of the SOVt'n mutants of Rhodotor1Lla rubra two had lost nllability to form {1-carotene, two remained ilpproximately the same as their parents, and threo had increased in their Itbility Lo form {1-caroteno as much as threefold oyor that of tho original stmin. Tn studying caro­ tene in Phycomyces and tho associated polyencs, Goodwin and Griffiths (45, 1.6) deemed it desirablo to study these compounds in morpho­ logical mutants of P. bZnkesleeanus and, in a second specirs, P. nitens. All cultures studied produced the Sfimc polyenes in the Sltme relativo Ilmoun ts. Carotene synthesis was similar in all - stmins; whel"C'ils the + straills produced only about one-hulf as much as the - strains, except for one stmin, which gave yi(1lds equal to that of the - st1'llins. FUNCTION" OF CAROTENE IN SEXUALITY OF :MUCORALES

In tuo order ~[ucornles two conditions r).ist for soxuftl reproduction. Tn some specie's, cultures started from a single asexllill spore will tthntys result in cui lures tlmt pI"Oduce ftsexual spores and, in addilion, form sexun.l spores (zygosporl's). These sl)('cies tHO dnsignat('(i Its homotlHdlic. In the nmjority of species, each will be mnd(' up of t,,·o forms and only two forms that, must b(' brought togethrr lIndl'l' aPPI"Opriate conditions ill order to forlll zygosporos. Such species m·o stlid to be hotorotlmllic. The two forms ,,-('re discoverrel in 1904 by Blflkl'slpp (10), who dpsig­ llated them as + or -. He drsignatpd t1w strains that grow Ipss lux­ uritwtly itS -- and tho more luxuriant growing str-nins as +. Tn 1920, Satina nnd Blakeslee (rJl)) statrci that thp f(,lllale stmins w('re + nnd thn male stmins We1"p -. Bin,krslre (11) notNI that when + n.nd ­ stmins of ciifl'err'l1t specips in the ~rlleomlos w('re placC'd tog-dhrr, nil imperf('ct s('xuairp{lctioJ1 look plllc('. In this plWnOlll<'IlOn zygosporps arp not mH.tur('d, for the imperf('ct rnllelioJ1 U5U111ly slop:i nl tht' progill11Pbll1giltl stngo, 01' mny oyrJ1 progress to fusion of gaml'tm;. 5SGGGSO--dl----2 8 'l'EClli~ICAL BULLETIN 1245, U.S. DEPT. OF AOfUCULTURE

This behn,vior in hC'tl.'l'othallic strains mndl.' it possible for Blakeslee (13) to nssign cOl'I'osponciing + and - signs to strains of othrr species. It also led him to the conclusion that thero must htl something funda­ mental and common to ILUmces of + stmins lwd likewise for all ­ strains of members of the :Mucorllies. In other words, all - st1'llins of ~Iucoralcs must have one 01' more chemicals in common which are chal'llctoristic of that mating typC'. Although it number of conditions were investiglltcd, no definite compound or chemical reaction was found to be charactm lstic of rither + or - stmins. Burgeti' (22) demonst1'lltcd that sex hormones were prrsont in tl1l' Mucorales, and rcc('ntly PIC'mpel (93) and Burgl.'ti' and Plompel (23) published further work on the hormones of l.iucoT' nnd Phycomyces. For a general discussion of the physiology of reproduction, sec Hawkrr (72). Lendner (82) believed thitt the yrllow pigment was ahntys regularly stored in larger amounts in the progametangia of + stmins than in the progametnngia of - strains. Sntina nnd Blakeslee (98) agreed after a study of numerous mating pitirs ill tho 1'Iucomcc·n.r.. TIH'Y brlievrd that the more pigmentrd strains (+ reactors) werc femalr and the ­ ]'ractors wrro male. With our brst .B-cllrotenr-producing st1'llins the + strain shows more pigment than tho - stmin. The same con­ clusion was held by Chodat and Schopfer (29) as it result of Iln intensive investigation of ]liucol' hiemalis. Schopfer (100, 101) stutrd titfl.t the composition of the medium and thl.' light affected carotene formation. He illuslmc('(l how tbe Cflrotene WilS conr~entrated in oil droplets in thc mycelium and even noted that. crystals of carotene wore seen in hyphnc of -nf. hiemalis. Un found that the + strain always had morn ClLl'otC'ne and a more rapid rllte of absorption of nutrients from the mC'Clium. In discussing the function of .B-carotencin sC'xtltlli ly of the ~ [UCOI'Il­ ceae, Burnctt (24) statrd that thn cvidence for it relationship brtwrC'1l carotene and sexual reproduction was poor. The cln.1ms of the rr­ lationship between carokne and srxual reproduction anI basnd on two facts: (1) Carotenes are found in gameUtngia during the scxunl process. (2) Different amour:s of carotene iU'C found in the mating strains. Burnett stfites that the second cltlim WIlS pl'ovrd falsr ns long ago as 1929 when Ling-Young (83) showed that in strains of ;"1. hiemalis which had high, intermediate, and low CfLrotcne contents, carotene content varied independently of thr nUtting type to which tlH' strain belonged. The observations of others, Ilccording to Burnett, were made on studies of too few strnins. Burnrtt studiC'cl the function of carotrnes with respect to number 1 abovr. He showNI that thc number of gametangia formcd were about thl' same when 11Splll'ltgine, glycine, or arnnl'mium acetate was incorpomted into the I11rdiulll, CAROTENOIDS L.'i THE FUNGI MUCORALES 9 although with ali1monium acetate ,B-carotene was reduced to a low level. However, only nbout half as many mn,ture zygospores were formed as were formed with the other two compounds. The second o).:perimentin\-olved buffered media, in which t.he I1lUnhl'r of game­ tangia, remained constant but carotene production was only lUllf itS great. Diphenylamine was used in a third experiment with the following results: Gametangia Zygotes Ill!. ,B-Carotellc produced matured (Goodwill) Diphenylamine (1:30,000)______284 197 25 Absent- ______282 182 62G Onp criticism of Burnett's work is that the author used data frolll Goodwin's experiments on carotene and compared it with progame­ bmgla and mature zygospores he obtained. The analysis for ,B-cnro­ tene should have been repeated with the same material. CAROTENE IN THE FAl\ULY CHOANEPHORACEAE Interestingly, Cunningham in 1895 observed the enhancpd forma­ tion of yellow pigment in C'hoanephora simsoni. Pigment in Choa­ nephoraceae was not investigated, aside from casual l'efel"Cnce to it, until studies were made by Barnett and Lilly. Since their publica­ tio.ns bear on production of inoculum for the ,B-carotene fermentntion, their investigations on C'hoanephorcL cucurbitarum nl"O briefly revie\\-NI. Barnett and Lilly (4) showed thllt the production of conidia in e. cucurbitarum was influenced by nutrients, by temperature, by light, and by aemtion. A temperature of 31°C. prevented conidia pro­ duction but permitted sporangia formntion. Good aeration was esp(~cially importlm t for the formation of conidia. Jn a second study Lhey (5) -reported that the nccumulntion of CO2 in 11. closl'd yessrl prevented sporulation. At t1. temperature of 25° C. or hig-lH'r, it relative humidity np!u' 100 prrcrnt rnhtlncrd sp0l"llngia production while lower retative humidities of 50 p('rcent fnvorrd conidii< forma­ tion. In a third pitper (6) they deseribN} the conditions affr(:ting the formation of zygospores in this species. Zygospores W(\1"r formed from 15° to 37° C. and were not Ilffected by light. Furtilprmore, zygospores would form in atmospheres conblining itS much IlS 10 percent of carbon dioxide. Stnrmtion of rnycPiium brought ahout zygospore formntion and this might occur hom Il pH 4.5 [0 S.5. They concluded tlmt the production of en.r·otrrH\ wu" stilllulntrd ill either culture by hormonc-likr substances or'iginnting from the mY('e­ limn of the opposite sex. 10 TECIL'Il"ICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE

In the same year Barnett, Lilly, and Kmuse (7) studied zygospore formation in liquid media. They reported that O. cucurbitarum pro­ duced zygospores in liquid media and identified the yellow pigment in this species as j3-carotene. They showed that when + and - stmins were mixed in liquid media, 15 to 20 times as much j3-carotene was formed per gram of mycelium as was formed by either mating type grown alone. \Vhen + and - stmills were gro'vn on opposite sides of a cellophane membmne, both strains usually increased pigment formation. This increase in color indicated that a stimulating sub­ !:ltance diffused through the membmne in both directions. They further concluded: ttlt seems probable that some relationship exists between the production of carotene anu sexual reproduction in O. cuc1lrbitarum." In contmst, Heichel and Wallis (96) reported that ",hen + and - strains of Phycomyc{'s were mated under the conditions used by Barnett, Lilly, and Krause, no enhanced j3-carotene yields could be obtained. Occurrence and History of the Choanepho!"aceae Since the question arises as to where members of the Choanephor­ aceae are isolated, it is appropriate here to discuss the records of their isolation from nature. Other ),.[ucorales known to form carotene arc each more or less ecologicfllly restricted. ]{ucor hiemalis is most often found in forest soil; Pilobolus is strictly coprophilous. 'rhe occurrence of Phycomyces has recently heen reviewed in detn:il hy Benjamin and Hesseltine (119). Cultures of "A1:ucor hiemcilis and Phycomyces species are n:vailable in many culture collections. How­ ever, members of the Choanephoraceae, especially m(Lting types, (Lre not as re(Ldily (Lv(Lilable, probnbly bec(Luse of the greater difficulty in obtaining material containing the org(Lnisms for isolation and ill m(Lint(Lining vigol"OUS pure cultures. The genus Choanephora was first described by Currey in 1873 (132) from fructifications on flowers of Ilibiscus rosac8inens1s fumishecl him by D. D. Cunninghitm from Calcutt(L, India. Currey describeu it first as Cunninghamia but later changed the gelleric name. to (llwanephora since the nflme Cunninglwmia bad already been used for a conifer. Cunningham (130) ["('ported in detllil on this species, O. infundibulifera, which he found growing in Ilibi.scus (Lnd Zinnia flowers. He recog­ nized the zygosporic stflge itS a sexual one, described it in det(Lil, and correctly placed it in the :Mucorules. He g(Lve tbe following tabular statement of the forms of reprocllJ(~tive bodies: 1. Sexual rcpl'oriucf ion ___ .. _ _ _ _ Zygosporcs IT. Asexual frllctifiClltioll ______~ . ___ . ~ _____ . _. _ Conidia, sporangial spores, chlalllydospores CAROTENOIDS IN THE FUNGI MUCORALES 11

In a second paper, Ounningham (131) described a second species, O. simsoni, including the zygosporic stage. Here he stated in con­ nection with zygospores: The colour of the contents of the conjugating processes varies from different shades of yellow to strong reddish, and not unfrequentIy, at a time when the con­ tents of one process have already been completely accumulated terminally and separated by the formation of a partition, those of the other are present through­ out its entire course and continuous with those of the parent mycelial filament. Thus at this early date the accumulation of carotene during sexual reproduction in Ohoanephoraceae was observed although the material was not iden tified. This species was found on leaves of Ipomoea rubro­ caerulea and Zinnia elegans at Oalcutta, India. Thaxter (178) named the species Choanephora cucurbitarum and re­ corded its occurrence on squash in wInssachusetts and on Hibiscus and a wild malvaceous phmt in Florida. Berkeley (120) reported this second species from putrid squashes sent to him from South 011,1'­ olina by Ravena1. Thaxter (178) further reported thttt :Morgan col­ lected it in Ohio and thttt n. specimen exists on squushes from North Onrolina in the Curtis Herbarium ttt Harvard. ?d611er (156) recorded the same species under another name on Hibiscus from Brazil. Peck (163, 164-) listed the same species on squashes from New York und(,1" the name Rhopalomyces cucurbitarum, as did Clinton (128) on squash from Oonnecticut. Rick (166) reported C. infnndibulifera from Bra­ zil. Sydow, Sydow, and Butler (175, 176) saw mllterinl of Choane­ phora from India. Thaxter (179) described the genus Blakeslea and the type species, B. trispora. He noted that the mycelium ",h(,11 first cultivated had protoplasm 'with fat globules and was bright orange-yellow, but upon continuous cultivation on potato agar this color b'Tadually faded to H light yellow. He found this organism as a contaminant with Botl'ytis from a larvae from cowpeas collected in Fioridi1. According to Thax­ ter, it probably was growing on the cowpelLS. Saito and N aganishi (168) found what they consickr('cl lo be fl new species of Cnnningha:mella, C'. mandshlll'ica, from uir over :\[anchurin, but this has been shown to be Choanephora manshul'ica. 'Wolf (187) reported O. c1wn7'bitarnm f!'Om squash, flowers of Cllctl[ll­ bel', FIibiscns s'ljriacns, II. coccineltS, okra, und cotton. He also described the zygosporic stage of this species. Furthermore, he showed that certain insects transmit the disease from one flower to another and that infection in squashes OCCllI'S through the flower end. This species was found by Dastur (138, 139) on chillies (C'ap­ sicum sp.) in India and by Wolf and Lehman (188) on cowpeas in North Oarolina. 12 TECfu~ICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE

Gandrup (14·4) and Ultee (181) studied Blakeslea trispora on freshly picked tobacco leaves in Sumatra. Palm and Jochems (162) reported C. c1LCurbitarum from Amaranthus blitum and A. spinosus. Burger (122) found a serious blossom blight of dahlias caused by C'hoanephora sp. in the Gainesville, Fla., area. In 1925, Choanephora persicar'ia was described by Eddy (141) on decaying peaches in the New York market. This speeies has never been reported again and was especially interesting because only sporangia were produced. The same year Couch (129) described still another new species from the flowers of Hibiscu.s 81Jriacus in North Carolina and noted that it failed to grow on squashes. A Choanephora sp. was the causal agent of leaf rot of ground nut (Arachis) in Batn,via, according t,o Van Hall (182). Jochems (148) reported C. inJundibuliJera on hibiscus flowers in the Dutch East Indies. Choanephora sp. was discovered at Buiten­ zorg, Java, as it secondary invader of plants of Lochnera rosea, which were infected with a species of Phytophthora according to Schwarz (169). Weber and Wolf (186) reported that their Blakeslea trispora cul­ tllTes from cucumber leaves were orange or yellow at the surface of the media on 2-percent potato dextrose agar. 'When their cultures were grown on 5-percent potn,to dextrose agar, the orange color was much intensified. They described the zygosporic stage of the genus. They also report that another strain (123) was isolated from a culture of fungus sclerotia by the senior author. According to Jochems (149), Blakeslea trispora commonly occurred in Java and SUIDatriL on faded leaves of tobacco, which had been attacked by the great, green tobacco bug (Nezara viridis), tLnd hence the fungus wa." con­ sidered a weak parasite. He also found it in drying sheds on new tobacco leaves. In addition, he found it on Physalis angulata, Ipomoea balalas, and probably other plants. Dade (134) reported that C. cucurbitarum occurred on papaw leaves, cacao husks, and withered blossoms of Hibiscus and Zephyranthes collected in the Gold Coast region of Africa. Blakeslee and others (121) stated that C. cucurbitarum is commonly found on withered flowers of pumpkin and squash from gardens near Cold Spring Harbor, N.Y. Stevens (173) found C. irifundibuliJera on petals of Hibiscus in British New Guiana. Wallace (184) recorded a Choanephora sp. on sorghum from TanganyikfL. Three species of Choanephora (C. cucurbital'um on leaves, stems, and flowers of Capsicum spp.; C. simsoni on Ipomoea rubro-caerulea, and on flowers of Zinnia elegans; n,nd C. irifundibuliJera on flowers of Hibiscus rosae-sinen,sis and on inflorescence of Tabernaemontana coronaria) were reported by Butler and Bisby (124, p. 8) in their monegraph of fungi in India. C. cucur­ CAROTENOIDS IN THE FUNGI MUCORALES 13 bitm'um occurred as a blossom blight of peppers in Florida accm.-ding to Weber (185). Tai (177) apparently found the same organism in China, although he called it C. man8hurica, growing on the flowers and pods of Dolichro8 lablab. Miyake and Ito (155) reported a disease of squashes caused by what appeared to be Choanephora., although they called it Choane­ phoroidea f,ucurbitae and stated it wns an Oomycete. Asuyama (116) also reported the same fungus in Jnpan as causing a soft rot of squash. Deighton (140) found Choanephora sp. attacking flowers of (lrotalaria 1'etU8a and clLUsing a die-back at the seat of infection. In .Malaya thc lower leaves of cassava were aHn,cked during wet weather by C. c'u,cur­ bitarum according to ThoIIIpson (180), and Su (174) reported the same fungus on chilli in Burma, Singh (170) isolated a strain of Choanephora sp, from Punjab soil, which he stated had not been previously reported from soil. Christen­ berry (126) found C. cucurbitarum on old flowers of Hibi8CU8 sp., Cucu­ mi8 sp., G088ypium sp., and Capsicum sp. and on the flowers and young fruit of Cucurbita pepo and Solanum melongena val'. e8culentum. It was the first report of this fungus on eggplant,. Lefebvre and Weimer (152) found C. cucurbitarum growing on cow­ peas in Georgia find st~ted that it is a saprophyte on the leaves of vll,rious grasses in Georgia and Floridl\.. Sinha (171) reported this species and Blakeslea trispora from Coloc(l.sia antiquo1"lun and C. cucur­ bitarum (172) as ctLusing a wet rot of chillies. Ohristenberry (127) isoln,ted B. trispora from flies and on leaves of Liriodendron t'lllipiJera ILnd C. cucurbitarum on eggplant flowers and fruit in North Carolina. ChoanephoT'a cucurbitarum was stnted to produce 11. necrosis of the cotyledons of marrow seedlings at Antibes (113). Karling (150) mentioned a Clwanephora sp. collected in Brazil, which had been iLttlwked by a chrytrid. Vestal (183) noted that low temperatures lLnd humidities from June to September at Allahabad, Tnelia, in 1945 reduced leaf-spot fungi white some saprophytic fungi, such as C. cucur­ bitarum, became abundant ererywhere. Liu (153) studying the fungi nssocinted with soybean seeds in Chinn. isolated {,hoanephora sp. Barnett and Lilly (117) reported Lhat one of their cultures of C. cu­ curbitarum was isoln,ted from squashes in West Virginia. Roger (167) recorded the occurrence of C. injwulibulifera on the flowers of Hi­ bisCU8 e8culenta. During periods of high humidity a blossom blight of dahlias eaused by C. injundibulijern o('clllTed in India according to Jain 11,nd Nema (147). Norton (161) reportl'd ('. cllc'l1rbitarum on squash in Texas. Ditntas ILnd Robeiro (137) encountered e. cucurbi­ tarnm in Brazil, and later Danhts (135) reported the. occurrence of C. conjuncta on fading flowers of fIibi.~cu.s rosae-sinensis in Brazil and 14 TECIThJ:CAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE noted that both conidia and sporangia were produced in abundance on oatmeal ttgar. For a more detailed account of the hosts of C. cucurbitarum and of O. injundibulijera, especially in South America, see Dantas (136). Hesseltine (145) monographed the family Choanephoraceae and included onl.y the genera Blakeslea and Ohoanephora, excluding other genera such as Cunningham ella , which had been included by Zycha (189) in the family. This same paper reported the occurrence of C. cuC'16rbitarum on C'ucurbita pepo in Wisconsin and on O. maxima in New York. In 1953, Ellis (142) noted the occurrence of C. cucurbit.arum on summer squash in North Carolina. The following year Ara and ~rahmud (115) reported the same fungus on Zanomia indica in India where it occun-ed as a blossom blight. Naganishi and Kawakami (158, 160) reported the isolation of Blakeslea trispora from the air from Japan. In addition they de­ scribed a new species, B. circinans, from pasture soil in Japan (159). In still another paper (157) they reported that Choanephora cucur­ bitarum and C. injundibulijera were isolated in Japan from faded flowers of dahlia, cosmos, squash, cucumber, canna, and morning glory; but it is not clear which species OCCUlTed on which bost. The most intensive attempt to isohtte strains of C. cucurbitaruTI1 was made by Poitms (165). He obtained this species from soil slLmples from Illinois, ~Iexico, Centml America, and the Islimel of Ponape. CheYltugeon (125) reported the occurrence of e. cucurbitar1L1n in the basin of the Cavally River (Ivory Coast) Africa. Baudin (118) also reported Choanephora [rom the I Yory Coast. Hesseltine and BeIl­ jamin (146) studied C. circinans stmins from soil samples from Trin­ idad, and from an isolate from a textile salllple from the Canal Zone. They also studied a strain of C. conjuncta from soil in Georgia, and n strain collected from air in Venezuela. Agnihotbrudu (114) reported isolating e. cucurbitarum from the rhizo:::phere of the pigeon pea «('ajamts cajan). Recently :Miller and others (154) reported finding O. conjuncta in cultivated soil and in forest soil in a study of soil fungi of Gcorgia. In a study of fungi associated with flowers and fruit of pickling cucumbcrs, Etchells and others (143) reported isola.ting four strains o[ C: cucurbitarum. Recent taxonomic accounts of the family hayc bcen published by Hesseltine (145), Dantas (136)) Poitras (165), and Hesscltine and Benjamin (146). Life Cycle To undcrstand carotene fcrmentation one must know something about the organisms involnd. The family Chofl.nephorllcelle is considcrcd to contain two gencra) Choanephora find Blakeslea (74); in each, spores, whcther conidia or sporangiospores, germinate to form CAROTENOIDS IN THE FUNGI MUCORALES 15 germ tubes that, develop illto much brnnched mycelitIDl. The bl'lll1ehed mycelia gl"OW mpidly in Ilnd on the surl'flee of nutrient agar. vVithin 2 or 3 days the surface of the agar is eO\'ereel, About, 48 hours after spore germination, conidiophorcs 01' spomngiophores arise and become bmnched at their apexes, These short bmn('hes, in turn, brnneh it second time and form small vesicl('s which then beill' either ('onidin, or sp0l'angioles over' their sUl'faces. In the genus Blakeslea the vesicles ben,r spomngioles which eon tilin rellltinly I'e\\' sp01'!lllgio­ spores within a hyaline membmne. In (,hoanephol'(l the spores are bome singly as conidi/l. This difference in spore I'ormlttioll is the genCL'ic chnmeteristic thltt distinguishes the two genera, Both conidin, Ilnd spomngiospores from the spomngioles are purple or bl'Own and nrc orten chameterized by minute mHrkinb"S in the 1'01'111 of longitudinal striations. At fIrst the fmiting heads of either conidia. or sporangioles are \\'hite, then they becomc mther brown, and finltlly nend,v hl/l('k. These I'l'lletiflelltions typically are formed next to the ('dge of tllC' culture dish, pl"OblLbl,r hecause the llutI'ients nre exh/lUsted or because of bettt'r aemtion there. About the stlme time the fruiting stl'Uctmes nre produced, thin, slightly ypUowish, tlerinl my('elium is fOI'med which later mlty almost completd,r obscUl'e the fruiting llC'ade. Fruiting is chl1meteristically restrieted to til(' first 3 or 4 tllLYS of ~l'Owth. ~I.rcelillm, both uerilllllnd subslml,e, is ('olu'se iU1d without Sf'pllttions. The substrate my('C'lium .is gcnemlly filled with ciropkls of ydlow ll1atNinl. In addition to the eonidiophol'l's 01' spol'ilngiophol'C's beltl'ing only spomngioles, It 5('('011(1 met hod of asexual l'(·prod udion iln-okC's tnH' sporangia. r1'l1('se may Iw I'ornwcl in variabh' numlwrs d('pC'neling on thC' species, the tempe1'!lturp, 01' nutric-nt ('onditions. TIll'spOl'llngio­ phon's, which likewise 11rise usually I'l'Om lh(· suhstrntp myc(·liulll, !U'P hYlllillt' but sometimes become slightly pignlC'ntecl abon'. In ttll e!1ses tht' sporangium is borne in It nodding 01' eireinnJe fasllion. Th.:' spoL'ltnginl \n111 is eh!lI'fld('l'izpd by being rein Ii \'('Iy I'esislan t bu t, ",hpn mature, by splitting into equnl hains. This ehlll'tl('lerisJi(' is Itlmost diagnostic of the fnmily. lnsidl' the sporllll~iulll 111'(' nunlt'rous spo­ rnngio;;port,s llnti a lypielllly pignwn ted eolulllella. whi('h lIllS it mon' or ll'ss distinc-t ('ollar attached Ilt its hllSl'. TIl(' spomll~iospon's I'l'Om spomll~itL ltl'e often pigmented and in all but 011(' spt·ci(·s Il:1n' IOIl~, stiff, hYltline appendages in tufts at both (·ntls and sometinH's ('\~('n nt it third 10clLtion. In addition to lIl('SI' types or IISt'XWtl spon's, hyphae in the substnlte mn)' round up into ilT<'gulnr ('(\lIs rC'po1'll'd. as ell l:unydospores. As the ('ulturp be('ol11('s older i[ take'S on H 11101'(, .\'!'lIo\\' lIPIWtll·aIH'(·. This is partinrla rly t rut' of J]/o!.-es!t (l Irisp(}l'fI. \\'hen held a ( room tempe'mturt' thl' ('ttlt 1l1'l'S rpmnin yinble for l'onsidemble periods of ..., 16 TECHNICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE time. However, cultures phtced in a refrigeru.tor show ermtic sun1vul. Lyophilized spores keep very well, provided that stocks are lyophil­ ized within it few days after the fruiting heads have become dark in color. Sexual reproduction is practically identical in all species of the family. .l\II species that have been studied are heterothallic, that is, the sexes are separate. To induce the sexunl stage, it + und - strilin are plnced a short distance from ead. other on nutrient ngar ilnd the colonies are allowed to come into contact with each other. As the colonies come into eontaet, the first notieeable gross ('ffed is the in­ creased intensity of yellow color on the reverse side of the colonic!:> where they meet. As the culture becomes oIdel·, this colol· beeomes a deeper yellow to yello\\~ orange. With the maturation of zygospores the yellow-orange color either disappears 01· is masked, and in its place nppears a dnrk zone caused by the hundreds of mature zygo­ spores. The development of zygospores, when studied miel'oscopienlly, hegins ns a coiling of filaments from each of the opposite m:lting strains. The twisted and irregularly coiled hyphae eyen ttmlly di­ \-erge llnd form swollen progametangia that resemble It pllir of tongs or It bow. Ench proglUuetangium forms a septum tllnt delimits lL gametangium which comes into contnct with its mate; the wnll be­ tween the gametangia breaks down and mixing of the cytoplasm results. Thus, the fused gametangiiL become the zygospol'e, \\-bich now enlnrges and assumes It more or less globose shape. The thie-k wall of the zygospore is minutely striate, but it neyer becomes rough­ ened with long project.ions as is seen in the zygospOl·es of mnny other genera of ~[ucol"l1les. The \ntll bee-omes it deep brown. At first the zygospore eontents show numerous oil droplets; these Intet· ('onlesee into one, (·,'nlml, oil droplet which is ycllow-ol"l1nge. The zygospores nrc alw!LYs formed eithet· on the surface of the ag!u· Ot· in the medium just belO\v the surface. The yellow-orn.nge oil droplets nrc numerous nnd ("oneentmted in the progalllctangin, gnme­ lnngin, and the hypillle immediately adjacent to the area where zygospol·es nrc bping formed. When most zygospores haye re(lched maturity, the ndjaeent hyphae and gametangia become almost emply and lose the large nceumuiations o[ yellow-omnge droplets. Cutter (JSS) described the !ludear behavior in B. t,.i.~pora. According to him, zygospores germinate after 3 mouths to form nn extensin, submerged, vegetati\'e mycelium. Kishol"C (U;l) studied the ('on­ ditions affecting zygospon' formation. Ont' intpn'sting dlarnet('ristic of th(' family ChOHIlC'pilorncl'Hl', also common to oUWI" ::\[llcorules, is tbe cOl'nocytic condition of the myc(·lium (n, condition in which man~' nlle\C'i nrc present in the CAROTENOIDS IN THE FUNGI MUCORALES 17 cytoplasm ,,,-ithout c('ll walls). En'n the gamctnngin, contain many nuclei in their cells. It is important to rem('mber that all as('xual spor('s contain mor(, than on(' nuclt'tls. \Vhctll('r it is possible to induce mutations giving larger yields of .a-caroten(' is not n.t all ('cdain. Pl'csumably, each spor(' would contnin various nucl('i with num('l'ous sets of the sam(' chromosom('s. An ('ntir('ly differ('nt sittilltion is found in many succ('ssfully mutated industrial organisms, in which the conidia poss('ss but one nucleus with one set of ehromosom('s. ~rembers of the Choan('phomc('a(' growing in nntur(' cnus(' n we!.. rot of flOW(,I'S or young fruit. Fndoubt('dly the conidia procluc('t\ on these parts ar(' cllrri('d by ins('cts from one plant to anoth('r. 'l'hf' inf('ction docs not appenr to b(' of nn~- pconomic importanc(', for uStmll~T only the blossoms aT(, attack('d. In some cns('s th(' mycelillm np­ parmtly ('ntl'I'S th(' fruit through tilt' blossom (,IHI a.nd caus('s thp fruit to turn y('llow, th(,11 hlllek nnd soft, However, no furth<'1' invasion of the host plant occms. Carotene in Choanephoraccae ~Iyfirst ncquuintanc(' with m('mb('l's of tlH' Chon.npphon1('('fl.(' began in 1946 ('1'3) with nstudyof ('hoa,nf'phol'(Lcu.cw·bilal'ulnoceurl'ing on pumpkin ill Wisconsin and of a stm.in of Hl(tkf'slea trispol'(L obtnin('d ft'om the Bnnrn ('olketion. Since it is my ellstom to stlldy nil ~Iucot'llles on a synthetic: medium contnining glucos(' and nspnmfrille (a m('dium known now to l'nhnncl' carotl'ne formntion), the cultul'(,s w(,l'e studied g1'o,,-ing on it. Th(' yellow pignwnt wns pronoUll(,Nl, especinlly in the B. trispora stmin. 'I'll(' taxonomy of the klll)\nt memh('t's of 1Il(' rn mily \\,itS t h(,ll dl'nloppd as flu' as possible. Thus nUll H'rs stood lin (il 1055, ,dlPll whnJ nppefired to Iw n /H':l"ly llonsporulnting culture of ('l/oa:11f']>lIo/'(£ ,nlS r('cpiv('d from til(' Qual'll'l'­ master Corps ('ollpct ion. It n,ppenl'('(l to 1)(' d ifl'(,l'l'n t from nil ot hrl' stmins sel'n, so it "'ns Pl'pSpl'Hd ill thr ARR CuLllln' COIlN'lioll rOl' possi ble fu lul'(, sl udy ns n, IH'\\' SI)('('it's. Sollwtinw lnl('r We' l'('('(,i n'd a cn.tnlogur from 011(' of lh(' .Tnpllnest' cultul'r eo]Jpetiolls whieh li,;tpd it SI)('ciC's (,:lllt'd HLaJ.-f's(.ea, circinnns. \Vt' \\Tolt' fol' lhp cullul'e. inquiring ,,-1101h('1' the' nnnw hnd ht'('11 publish('d. In cltH' tillw WI' I'('ce'i\-ed a rpply sln.lillg that till' orgnnism hnd ill fuet I>('('n dr,;(,l'ibrd in 19;'55 and thn.t the sp('cies \\'ns bnsC'd on H. singlC' Rtrn.in. \\-Ill'n the culture nerin'd, \n~ w{'r(' intI'J'l'Rl<'d to find tllll! it npPNlI'C'd it\('nticnl \\'ith lh(' cultul'l' from Pnnnmu, which cnm(' to us I"rolll til(' Qunri.prmnstpl' COI'PS Co\lpct ion. Bot h sll'n ills pl'Od 11('('(\ ollly spo­ mngin. Since all otll('1' fOl'ms of Hla,h-ts/('n :illd ('ho(J.nrphol'((, l'x(,l'pl on(', possessed n. s('cond IllNlnS of ILs('xunl l'l'pl'Oduetion, ,,-(' \\"('n~ doubti'1I1 if cith(,I' s(rnin \\'ns typiC'nl or lh(' organism in Iln.lun'. Ful'­ th('1'mol'(', ",h('ll thpse cultun's \H'I'(, mn«'d Ilothing hn.plwll('d. \rl', 18 TECI-IXlCAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE

therefore, flgnill dropped OUl" work Oil these forms becfluse it appeared we had too little material with which to draw allY conclusions. In the mellntime, we had nssemb1ed it number of cultures in the family, at 1ellst one of which we had not seen before, namely C. C07l­ :iullcta Couch. The vnlidit}- of this species had been questioned by Poitrns (165). From two soil sHmples collected in Trinidnd, we ob­ SPITed an nbumhll1ce of sporilllgia of it ('hoanephora which, when isolnted, appeared identical with the lllnterial of B. circinans and furthl'l'l1lOl"(' sporulated very poorly in culture. On the same soil jsolation plates. numerous zygosporE'S occnrred thnt were typicnl of Cbonnephoraceae. Aftel" we sc1ected and paired a number of single sporanginl isohltes, - nnd + strains were secllred llnd these were found to milte with thc Panamn and Jnpanese stnlins. It sePlllNl, tlH'rdoI'e. highly desirnble to report the OCCUl'l'ence of this species sinel' \\'e had zygospores, and to place it .in (1wuneplwra whet'l' :lnotlH'r sp('cies, ('. pel'8ica.l'ia Eddy, hnd :ll!'elldy been described with only sporangin. At the same time, we lI!lel accumulated 11 lllunl)('r of stmins that could not he determined IlS to known sp('cies because of their poor spol'ula lion. H Cllce nllmerous m:lting cxpe-l'imen ts between e. circinalls (Blakf8lea cil'cincws) , B. fl',zspora, and e. CllC11l'bital'wn \n'l'(' made in ordt,l' to idrntUy our strnins eOlTectly (77L \rlli'll it IlH'llllwr of one species was milted with :l strain of the opposite mating type of :l difl'el'('nl specit,s, the (\-pical .\-eHow 7.one nppear('(1 on the renorse' sidt' of the plate, but it failc'd to nppl'ar if the stmins wel'l.' of the same mating re!lction. Fmthel'lllOre, the ~-ello\,- ZOlle persisted if till' slrnills W('I'(, of difl'en'll t speeies; \\"Iwl'ens, if t he,'- \\-ere of the same specips, the' yellow zone usuHlly disappe:ll'('d IlS zygospores matured. In imperff'd l"f'aetions nOlle or onl~- H fe\\" nbnonnal zygospol'es "'C'n' fo rlYl (·d. Xagnnishi fI.lld Knwfllmmi (rJO) had l'epol"t('d on the morpholog.\" of (he' sexunl organs in impNfecl I'l'Hctions be-tw('e!1 spt'cies of the Cholll1P­ pitOl'!lCelll'. 'Ye concludc'd tlult if one had mating t.\-pes in ont· species in the fllmily ChoHlIephomc('tH', the mating l"l'llction of any specie's ill t'ithpl' genpl'!l could be dett'l'mi])e'd h.,- the- illcn'/lS(' oI-JllCk of incr('ase ill yt'1l0\\- pigmentation on the re\-l'I'Se side of lllilted colonies Ilt t Iwir point of lIllioll. Ahout this time we became interestcd in a report (,hat zygospores ('ould he produced in liquid media in shuke cultmes. This OhSeI'Y!l­ tion may bel of some significnnec in tbe c\'olulion of the ~[ll(,ol'illesJ sinN' ()lJC' of thp postulatl\d lln('estors is the Snprokgninles 01' water molds \,-hose' s('xuill spore', oospore, is fOlllld ill aqueous medin. Ae­ cOl'llingly, we llsedlllllting types or n, numlll'r or geIH'l'!1 of :\[ucomles, gro\\'ing tite com billed JIlating types ill s,nlliteti(' liquid mediuJll und potnto drxU'OsC' hroth. Till' Inli(,I' medium wilh ng-nr is ('sp('ci!dly CAROTENOIDS IN THE FUNGI J\iUCORALES 19 suiblble for zygospore formation. In most of these experiments we obtained negntiye results, but we readily confimled Barnett, Lilly, and Krnuse's report (7) regarding C'hoanephora c1lc1lrbitarllln nnd showed in the two other species of C'hoamphora ancl the one species of Blakeslea that zygospores were rOI'med readily on potato dexlrose media in shaken flasks. However, no zygospores could be found e\'en though good vegetative growth resulted on the synthetic medium composed of glucose, aspamgine, salts, and thiamine. The appen,mnce of shaken cultures grown in the synthetic medium nJtel' 72- hours nt 28° C. on 11 rotary shaker was very striking (75). The mycelium from the flasks ill which Lhe + and - strains had been inoculated appeared as large, yellow-omnge-pigmented balls resem­ bling canned yellow peaches. The actual amount of myeelium from the flasks whcre + iLnd - slmins had been grown Wl1S approximately the snmG as when either stmin WitS grown by itself. The single strain eultures lneked pigm('ntation. Dr. R. F. Anderson and asso­ ciates Ilt our labomtory ran .a-carotene annlysis on the mtlterilll fl"Om this lu)(l other experiments Ilnd sho,,'ed that the mllted cultures contained pigment composed of Ilbollt 80 percent .a-carotene according to the A.O.A.C. method. In It11 [our species, Blaf..;poSlpa tri8]J()t(l. (,hoalU'phoNL circinans, e. cOlljunctCL, and C. CllcllrbitCLI'Il7n, the yiC'ld of .a-c!1l"otene in mn.t('cl fermen talions was H,t lenst double that obtain('d with n. single stl"llill gro\\-n alone. Inter('stingly, the B. tri­ s]Jora strains XRRL 2457 (-) and XRRL 2456 (+) each gilye higher ....ields of .a-carotene than the mating types of :U1Y otbet· sp('eies. '1'ypiC'ltl results 11re sho\\-n for C. circinans on the syn Lhetic medium.

Dry l("cight {3-Carotenc Strain: g.'SOO mi. '/'0/(1/ /lgm /l(J1n g. ;';-RRL 25·16(+) ______O. 82 20 Xn.RL 25-18(-) __ . ______. HI) ;)5 50 XRRL 25·1G X 25-18 ______.75 'Y­_Ou 3·10

As Iloted, whell strains of opposite mating type's from difrcrent Sl)('­ cit'S or species from difr('rent g('nera arc mated, the yellow color ap­ pears aL the juncture of lhe colonies.\Vh('n such strains arc mated in shake cultures on synthetic media, typical resuits slich as til(' fol­ lowing arc obtained: Dry weight {3-Carolene Strain: g./SOO ml. TO/'Ll /lgln p.gmlg. B. trispora (-) ______• ______0.58 no 155 C. ('onj uncla (+) _ . _ . ______.71 18 25 13. trispora (-) X C. conjnnc/II (+) __ .n 212 2!JO Thlls til(' same (,l1hn11 c('(\ produc'liol1 of .a-e:ll"O\('I1(' ean J)(' obtainpd. in int('rsp('cific crosse's as w('ll us ill inlmsl)('("ifi(" Cl"O$:ws. Although 20 TECR,"HCAL BULLETIN 1245, U.S. DEI'T. OF AGRICULTURE yields formed were not high as compared with yields of ,a-carotene from Phycomyces of 2,500 to 3,500 Ilgm/g. reported by Nakayama and others (91), it still looked promising to study this enhanced carotene production with various conditions and media. In this work, reported by Anderson and others (2, 3), pieces of mycelium were taken from the stock cultures to start inoculum on a glucose­ asparagine agar (fig. 1). Each strain was grown separately. After the inoculum slants had been grown for 5 to 6 days at 28° C., the

NRRl 24S6 1+1 S••cks .n p..... de••r.s. .g.r I ~ NRRl 2457 (-) ~ ~ Glucose esparagile 5-6 ddYs ~ ~ 28·C. ~ ~ YdrCIIYled corn and casein 100 ml 8 e------48 hours at 28·C. ~ t Blendor 5 ml Blendor 5 ml inoculum ~~ NRRL 2456 x 2457 Vegetable oil (2 percent cottonseed 2 perconf soybeal) g 0.12 percent nonionic detergent {3 -ionone (sterile filtered) ~ added 2 days after Steamed 10-15 minutes inoculation ~ Solids removed, filter,ed and dried at SO-SS·c. .

FIGURE 1.-0utlille of method used for inoculating Basks for thc production of /3-carotclle. CAROT.ENOIDS IN THE FUNGI MUCORALES 21 entire mycelium was scraped off into a 500-ml. Erlenmeyer flask containing 100 ml. of the basal medium composed of: Grams per Wer Corn (acid hydrolyze.!) ______75.0 Casein (acid hydrolyzed) . ______2.0 Corn steep liquor______. ______5. 0 Potassium phosphate monobasic______.5 Mliligrams per liler ThiamiJle' hydrochloride __ . . • _ • _. ______.______1.0 pH adjusted to 0.2 with sodium hydroxide.

The corn was hydrolyzed by lluto('ill\ring" I\, 15-pel'eent slurry in 0.2 N sulfuric acid at 121 0 C. for 00 minutes. The inoculum usually grew as lnrge masses of mycelium which was macerated in a 'Waring Blen­ dol'. 3 Five mL of this material from each mating type was then used to inoculate 100 ml. of fermentation medium. The basal fermenttl­ tion medium as described contained the following additional adjuncts: 4 percent vegetable oil (2 percent cottonseed and 2 percent soybean), 0.1 percent ,8-ionone, ulld 0.12 percent nonionic detergent. The func­ tion of the detergent is to emulsify the oil, thus making it more avail­ able to the mold mycelium. The ,8-iollone was Seitz filtered, sterilized, and added after 2 days of fermentation. Following 6 days of fer­ mentation, flusks were steamed for 10 to 15 minutes to prevent enzymes from breaking down the carotene. The solids were removed by filtration fwd were dried at 50° to 55 0 C. in a vacuum oven. The analytical methods employed we::....: those described by Anderson and associates (2,3). 1Vhen the mated cultures were grown on the basal medium, a fourfold or fivefold increase in total yield of carotene was obtained. From preliminary work, B. trispor(J, appeared to be the best culture. The addition of vegetable oils incrensed the yield two­ fold. 1Vhen ,8-ionone, representing a part of the ,8-cnrotene molecule, was added to the basnl medium a fivefold increase Tesulted. 1Vhen all three adjunets-vegetnble oil, detergent, and ,8"'iol1one were added, n sixfold to sevenfold inerease ovel' the bnsitl medium wns obtn,ined. When othel' substances, such as 2-4 dinitrophenol, lipoic aeid, chloro­ fOTm, yeast-extract, liver-cxtrud, and potato-extrae.t were n.dded, no increase in ,8-carotene occurred. Intcrspedfie miLtings within thc four species glwe enhlll1('ed Cttl"Otl'l1e produdion, hut r('sulls are no bettcr than with B. tn~~7>ot(L NRHL 245(jX2457. On analysis, 75 percent of tbe tolal pigmcnt wns trans-,8-cllrotcne. When biologicnlly assl~yed in rats, the dried solids showed tbe ,8-caro­

3 l\Iention of prodUcts does not imply endorsement or recommendation by the Department of Agriculture over other products of a similttr nature not mentioned. 22 TECHNICAL BULLETIN 1245, U.S. DEPT. OF AGRICULTURE tene was Ilvnilnble fiS 11 precursor of vi.tamin A. Anlllysis of the dried solids gave the following: Percent l\J:oisturc. ______5.0- 6.2 A,;h______3.0- 3.2 Fat______52.2 -53.0 Fiber___ . ______5.2- 5.5 Total nitrogcn______.___ 5.15- 5.35 Esscntial amino acids wcre: Lysinc. ______2. 5 "l'IIdhioninc______. 4 In the feJmentation no zygospores were formed Ilnd pigment oc­ curred through the mycelial growth. Anderson et at. state that the formation of zygospores is not necessary for the incI'eased Ilmounts of cnrotellc obserycd. Further pllpers in the series from our laboratory (30,32) wel'e con­ cerned with tbe effect of various grains on the production of f-rllrotene by 1l1ated strnins of Blakeslea il'ispora. The grnins and soybean pro­ ducts were hydrolyzed and incorpomted into the fermentation me­ dium at tbe mte of 7.5 percent. B. tl'i,<;pora does not Ilppenr to hydro­ lyze stnrch. Acid-hydrolyzed, hexane-extmcted, soybean oil meal gn ve the highest yields of jJ-clU'otene, as well 113 the highest amoun ts of dry solids. This material was better than ground whole soya, which suggested that the process of extmction favombly nlters the mellI. Of the various treated soybelln meals, the hexane-extmcted soybean oil meal \\~as best. Furthermore, there wns little difference in concentmtion between 3 to 10 percent, illthough there is consider­ able \rariation in growth as measured by dry weight of solids. Use of' bexllne-extrncted soybelln oil meal with other 1l1ellls or gmins re­ sulted in no increase in yields. Best yields were obtained from soybean oil meal alone. A study or the effects of carbohydl'lltes on the fermentlltioll using 5 percent l'llrbohydmte ilnd 0.2 percent acid-hydJ'olyzed casein was made. This study showed thai dextrin Ilnd sucrose gn\'(' the highrst yields. "When these clll'bohydrates were added to soybellIl oil mellI, 110 material change occurred. Analysis of the pigments showed 05 percent was trans-jJ-carotene. The J'emaining fruction contained seven pigments. Study of the time course of the fermentation showcd that the pH did not fall below 5.9 and thnt mtlximum earotrne pro­ duction occUl'l'ed nt 5 or 6 days. When the + Ilnd - stJ'ains wCI'e mated 3 dRYS prior to inoculation, the incr<:nse in jJ-carotenc was not significllnt. The mycelium in these fermentations sho"..-ed distorted growth with lllrge irrrgulnr swellings with the pigmrnt unirormly dis­ pel·sed. In some, a red crysttllline precipitate that resembled carotene WIlS seen in the mycelium. CARO'l'ENOIDS IN THE FUNGI ::MUCORALES 23

Since vegetable oils enhanced production of ,B-carotene in B. tl'i­ spora, additional work was done on the effect of fats and oils on the fermentation (31,33). Each fat was added at the rate of 40 ml. per liter of media. Of the 19 oils tested, all but one greatly stimulated growth and ,B-carotene production. Growth, as measured by dry weight, ,vas greatest with peanut oil, but the most ,B-carotene WitS formed with white grease. Oils and fats that contain large amounts of oleic and linoleic acids, such as cot tonseed Imd soybeall oils or whi te and brown grease, produce thelargest,B-cnrotene yields. \Yhen whi te grcllse was used, dl·Y ,,{eight increased with increase inllInOunts of grease to 8 percent, but maximum ,B-cnrotene per gmm of dried solids wns found at 4 percent. Fatty acids were not as effective in stimulating ,B-citrotene formation as neutml oils and fats. l\.nalysis of (II·ied mycelium from these experiments did not indicnte allY shift ill the carotene pig­ n,ents. Thus, iralls-,B-cnrotene constituted nbout 95 percent. The third ingredient that stimulated ,B-carotene formatioll WilS ,B-ionolle. Cal"Otene synthesis increased with greater C011cen­ tmtiolls of ,B-iollolle, but the dry weight of the fermentntions became progressively less. To obtain Illilximum stilllulntioll, the ,B-ionone had to be added on the second day. If it were added at the st:lrt of the fermentation, a I-day lng in growth initiation o("C'uITed. The efrect was no difl'erent whell a-ionone was used. The use of a detergent (Triton X-IOO) had two effects: (1) it emul­ sified the fat, making it morc availil.ble to the mold, and (2) it brought about a dispersed growth of mycelia, a condition necessary for high yields of carotene. Other surface detergents were tried, but the b(>st results were obtained "'ith Tritons 45 through 100. Patents covering thr proC'ess of Illilking ,B-C'arotene b.,- combining + and - stmins of the Chollnephomeeae have r('('enUy bren issued (J ,3_i,76).

LITERATURE CITED

Carotene in LVlllcorales

(1) A:-

(5) BARNETT, H. L., and LILLY, V. G. 1955. THE EFFECTS OF HU~nDITY, TEMPERATURE, AND CARBON DIOXIDE ON SPORULATION OF CHOANEPHORA. CUCURBITARUlf. Myco­ logia 47: 26-29. (6) --- and LILLY, V. G. 1956. FACTORS A.FFECTING THE PRODUCTION OF ZYGOSPORES BY CHOAN­ EPHORA CUCURBITARUll. Mycologia 48: 617-627. (7) --- LILLY, Y. G., and KRAUSE, R. F. 1956. INCREASED PRODUCTION OF CAROTENE BY MIXED + AND - CULTURES OF CHOANEPI!ORA CUCURBITARUlf. Science 123: 141. (8) BAUERNFEIND, J. C., SlIITH, E. G., und BUNNELL, R. H. 1958. COLORING FAT-BASE FOODS WITH ,8-CAROTENE. Food Techno!. 12: 527-535. (9) BERNHARD, K., und AI,BRECHT, H. 1948. DIE LIPIlJE AUS PIIYCOllYCES BLAKESLEEANUS. Helvetica Chim. Acta 31: 977-988. (10) BLAKESLEE, A. F. 1904. ZYGOSPORE FORMATION A SEXUAL PROCESS. Science 19: 864-866. (11) 1913. A POSSIBLE MEANS OF IDENTIFY~NG THE SEX OF (+) .... NIl (-) RACES IN THE lIUCORS. Scicnce 37: 880-881. (12) 1920. MUTATIONS IN lIUCORS. Jour. Hered. 11: 278-284. (13) 1920. SEXUALITY IN ~IUCORS. Science 51: 375-382, 403-409. (14) BONNER, J., SANDOVAL, A., TANG, Y. 'V., and ZECHlIEISTER. L. 1946. CHANGES IN POLYENE SYNTIIESIS INDUCED BY llUTATION IN A RED YEAST (RHODOTORULA RUBRA). Arch. Biochem. 10: 113-123. (15) BRAITHWAITE, G. D., und GOODWIN, T. 'V. 1957. ~IEVALONIC ACID AND CAROT~]NOGENESIS IN PHYCOllYCES BLAK~Ji;- -.. -~~--,~ ­ LEEANUS. Biochem. Soc. Proc. 66: 31P-32P. (16) --- and GOODWIN, T. 'V. 1957. BIOSYNTHESIS OF ,8-CAROTENE FRO~I DL-,8-HYDROXY-,8-lIETIIYI,-O [2-cI41 VALEROLACTONE BY PIIYCO.\[YCES BI,,\KESLEEANl!S ANI> CARROT SLICES. Biochelll. Soc. Proc. 67: 13P. (17) BRUCKER, W. 1956. VERGLEICHENDE UNTERSUCHUNG DER PHENOL-UloiD KAROTINOlll­ lJIL~UNG DURCH PHYCOllYCES BLAKESLEEANUS. Die Naturw. 19: 450. (18) BUNNING, E. 1937. PHOTOTROPISllUS UloiD CAROTINOlDE. I. PHOTOTROI'ISCIIEWIRK­ SAlIKEIT VON STRAIIl,EN YERSC'HfEIlENER WELI,ENI,XNGE UND STRAHLIJNGS-ABSORI"I'IN IN PIGllENT BEl I'ILOBOI,US. Planta 26: 71.9-736. (19) 1937. PIlOTOTROPIS~IUS UNIl CAROTINOIIJE. n. DAS CARO'rIN DER REIZAUFNAI[lIE7.0NEN VON 1'1l,0BOI,l'S, PIIYCO.\I1'CES UloiD AYENA. Planta 27: 148-158. (20) 1937. PIiOTOTROPISlIJ;S UloiD CAROTINOIllE. Ill. WElTER UNTERlilf­ CHCNGEN AN PILZEN UloiD 1I0llEREN PFANZEN. Planta 27: 583-610. (21) BUNNELIJ, R. H., DRlSCOlJI" 'Y., und BAUERNFEIND, J. C. 1958. COLORING WATER-BASE FOODS WITH ,8-CAROT::NE. Food Tech­ nol. 12: 536-54l. (22) BCRGEFF, H. 1924. UNTERSCCIICNGEN UBER SEXl~ALITXT CNn PARASITIS~IUS BEl lll·CORINEEN. Bot. Abhundl. 4: 1-135. (23) --- and PLElIPEL, .M. 1956. ZUR KE~NTNIS DER SEXl'ALSTOFFE BEl lICCORINEEN. Die Nuturw. 43: 473-474. CAROTENOIDS IN THE FUNGI MUCORALES 25

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(41) GOODWIN, T. W. 1951. THE MINOR CAROTENOID COMPONENTS OF THE FUNGUS PHYCOYY­ CES BLAKESLEEANUS. Bioehem. SOC. Proe. 49: XXIII. (42) 1952. STUDIES IN CAROTENOGENESIS. 3. IDENTIFICATION OF THE MINOR POLYENE COMPONENTS OF THE FUNGUS PHYCOMYCES BLAKES- LEEANUS AND A STUDY OF THEIR SYNTHESIS UNDER VARIOUS CUL­ TURAL CONDITIONS. Bioehem. Jour. 50: 550-55S. (43) 1952. FUNGAL CAROTENOIDS. Bot. Rev. 18:. 291-316. (44) 1954. CAROTENOIDS, THEIR COMPARATIVE BIOCHEMISTRY. 356 pp. New York. (45) --- !Llld GRIFFITHS, L. A. 1952. CARo'rENE SYNTHESIS OF SOME NATURAT,LY OCCURRING MUTANTS OF PHYCO:lIYCES BLAKESLEEANUS AND BY PHYCOM\'CES NTTENS: INHIBITION OF CAROTENOGENE";[S BY STREPTOMYCIN. Bioehern. Soc. Proe. 51: XXXIII. (46) --- and GRIFFITHS, L. A. 1952. STUDIES IN CAROTENOGENESIS. 5. CAROTENE PRODUCTION BY VARIOUS MUTANTS OF PHYCOMYCES BI,AKESI,EEANUS AND PHYCO­ :lIYCES NITENS. Bioehem. JOllr. 52: 499-501. (47) --- GHIFFITHS, L. A., JAMIKORN, M., and WILr,MER, J. S. 1952. CAROTENOGENESIS IN PHYCO.\IYCES BI,,\KESLEEANUS. 2d Congo Internatl. Bioehim., PariS, AbH. J 952: 210. (48) --- GRIFFITHS, L. A., and Moor, V. V. 1956. STUDIES IN CAROTENOGENESIS. 16. THE ACTION OF SOME ANTI­ BIOTICS, ESPECIALLY STREPTOMYCIN, ON CAROTENOGENESIS IN PHYCOMYCES BLAKESLEEA:

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