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lAD

TECHNICAL MANUSCRIPT 532

0 PRODUCTION OF ETHYLENE

BY PEN ICLLU CYCLOPIUM

to . CEU -,e

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0OC E %a. OV LIC

I- 1 4-

Josoph Lonski E

Harry E. Gahagan III a

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APRIL 1969 C X -0'0-6 Ef 3.~ .. i u

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*4*4***44DEPARTMENT OF THE ARMY

Fort Detrick c

P, Do CO

Frederick, Maryland . .

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TI WHITE SrCTIlONDOc iIFF SECTION

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DEPARTMENT OF THE ARMYFort Detrick

Frederick, Maryland 21701

TECHNICAL MANUSCRIPT 532

PRODUCTION OF ETHYLENE BY

PENICILLIUM. LYCLOPIUM

Joseph Lonski

Harry E. Gahagan III

Plant Physiology Divis ion

PLANT SCIENCES LABORATORIES

Project 1B562602A061 Arl16

1

A RSTRACT

FtI lIV 14-11. 1, v od % tt Itill by v Volt ci uny I i tl j.& p jji un ATICC No~ hi I 'i wa

st utitid ill14 ieIitI'll ( 0 t tic I ift. eve It, tit' t lit ftuiil t.lei-.0. O's 111. wit s tt pr odite lid it I 1 [~ ot ti t k h cIs I tit t.v I.t .e r .1ie d i I -

1Th1X I Iitill diii ~'we -I l . t1411 i lte i' t*0 e v~t~l. t ra l i tl of ii 0 tit Imed i urwas4 vaied Ml. eiliine W.1 propor I I it'lil titI glwt i. Iloweve i. *whitl thleAI'i iouriti1t ratiectcil l o WasI varlt-1 d Or tilt litt rogt n 1o1irC4C lia I igod * luIItcIra w.tu ~I1 no ii' I' 1 re U a v t it u-11 I I I n c Ii mI t I Ill stZA I Si 0I tMA I nencedli I t'Iit. pIuuxtct it'l. hlatioi nitukettd I t ismila ted totalti Iy 10110 111,duct it'll; d ilutdoilte wasi . good t l!it I'l hi t it' tit'i titIrOtii I I ll.

Tht. s -i nt t~l't Of tile nlat Ilra I IV prodilcod ethylenet wasa ver ift ledbyv c'nvetit ligi~ t to I *2-dibromotOlaie 1111i ?howilig t hat Its aInfrared

Ci tee tu t a IMW1.41idt e I I o tI lat I t at t i I '- tItr onuot t1.

II NTROIUCT I ON*

lit I 'I. * oauy:. I'iit andd Mlte chemicallyI idelritIflmd a itymioalogied Ilvati ve' gase aminat Ii'it of Pu'tici' I ititm.dlI!it y as othylene Ivy preplrat Itillof Chia I er ival ives1 I..Jn 411t0111A t iigrapph ic ei'vdence anud lieoasaysv

"ir :ing p tdi; nui i lIs W ud Itv e that a ;tint'er ti' othiier ftnip myvt prodatieV1 hVlItite 4~

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tvc~~JI in (i) thie relat Ittd i p oft tit predacllt Iit'll Of ;'thbyIVce to' til, li ec ofIca' t he tI nugn at; .and ( I I I ) si't.e t acl t s -I t fec I it ti he' prodtnt I tit of*

ItI . mVnKR IAI.S ANtD r*UrttuS

The I sol ate s if I'vi c iI 1 n eve I apI int usoi 1' Iit theet stt id Iet was ATCL'No'. /t'i l I ba ined t ra~tn tilt- Amer icani Type Cu It tire Colle ictionea. St tickct itnes were nutIitt a inad en steile pot atoa-d41xtri'e agar Ill tilt, dark at"I I . C. Inot'caa 1 l ast prepared framn ;-clay-o!4 cat iI irs 'vy sa:king the

sportes into a~ f lank o'f stileI distitlled wat ci. A DeVI tiss atomnizecr Na'.I *'. IllittedII a will: a coll1 a r a retinal (hei' ita'preve'ntetitltta( (e etac pe Of

:aa'ctI ttn. wa:. nsed (oi' na'c IlaIa the f lasaks. ach f lask ca'n(;a inedapproinia'viv .40, 000 savt's Alter I itaca I t nte en Itutre: wort-N g ppi'i-a'a Willi catll e p Intgs. All iii IIt tia' Wee cI VOi'I il the diark i

."%, C atter ptl-' I (till nar e* x per tien ts intd icate a'I ta dli fference beta eweent dark -

.And I i gilt -grown cuit iresM

Toe sI 11ti the relatitonshaip et ethy'I e It, the tile evce of ethet fmuns,lw a CIt uins's Were %zrevuI Int "0-in1 I KxIetainey~ Oil " I ss t 'inl tit teriti

mledt i urn. The'ba sal cit re ined I uat hadl (lit- I ciI awInte comapos itioIn: t te ros e., Nit, NOl j.*.. U'O.. l. 0 . ;io, 1.. e0, :1 . .. ' ;lilt .cc g .e 1.0 m[t %It atlcctint 0 s'iiti'it (siled tii lt olowtngIIadI c ,'aac ent ra t i cuts Ot ( tic, a, 1 4-11101 t% t pOr I iI tOr : a ('11 .().) 11g. .'t. 0.0" SSW,0.0I 111)1W.; Mit, 0."% nig; antd tIl. 0.'1 Iitg). And radlistilled wale ts n 'ikka

I Itit e . Trhe~ Jill a'l t he iteltin a Si.ad - lus-.t ed ( o a'1 "0 pr tI'll I l It- Inv i tgdudi wit,*. tntudl i t i be - .84 il Iet, .I c ta Liin . Th I - was l.iit I te p lit t it:I Jiranige till' ci Iit i

STh is aripo'rt slti' idt'!~ ba' at sed u . Itt a'trd ut ra c iI l a lit in I'terial I I e l'epa111th I sed inl i lt , Ope e n It erat tir a*. Reddelr -. i tat i'r01 t i'd il t et 41111ac luSg tlita,i lt orlial I $Ia Ci'nt a1 I14dt hll*,t Iair t alin II v cati1ct [ heI. selllIc tlt h'i t Ia dace td l11itWlaa'1 atd Wheat 4,f Ina v~ V ppet U i it . I I a It I e t 01111t

4

Every 8 hours the cotton plugs in each of five different flasks werereplaced with sterile rubber vaccine stoppers. After 8 hours the ethylene,carbon dioxide, and oxygen content of the air space in the stuppered flaskswas measured. Each value reported is the average of four replicates.

In the series of experiments in which ethylene production from variousmetabolites was studied, each value reported is the average of four replicatesof three flas'.: each. Different 50-ml Erlenmeyer flasks were stoppered every12 or 24 hours, and the ethylene and dry weight were measured.

The gases were measured with an F&M Scientific Corporation (Model 720)gas chromatograph.0 A flame ionizintion detector (hydrogen-oxygen flame)was used for ethylene and a thermal conductivity detector (175 ma) wasused for carbon dioxide and oxygen. Columns used were molecular sieve toroxygen, silica gel for carbon dioxide, and activated alumina for ethylene.'

After the gas composition was determined, the flasks were warmed tomelt the agar and the contents were filtered with suction on a BUchner funnel,using tared filter paper. The filtrate was retained for pH determination.The fungal mat was then washed with three 10-ml portions of hot distilledwater. The filter papers and fungal mats were dried for 24 hours at 80 Cand weighed to 0.1 mg.

To prepare the ethylene d rivative 1,2-dibromoethane, cultures weregrown in nine Fernbach flasks on 500 ml of sterile basal medium, supplementedwith 47 asparagine per liter. After the cultures began producing ethylene,the cotton plugs were replaced with two-hole rubber stoppers arranged sothat air could be swept through the flask. The air stream was passed througheach flask for 30 minutes on four consecutive days, bubbled through 150 mlof 0.5% Br in CCI 4 , and finally bubbled through 100 ml of a 1% solutionof NaSC 3 to trap any vaporized bromine. Then the CCI4 was equilibrated withthree 50-ml portions of 0.2 N NaO 4 to remove the unreacted bromine. Afterdrying overnight with 20 g anhydrous CaSe4, the CCI4 was reduced to a volumeof 2 ml with a rotary flash evaporator - was reduced further under a streamof nitrogen.

The 1,2-dibromoethane synthesized from naturally produced ethylene andauthentic 1,2-dibromoethane* were chromatogr phed with a Perkin-Elmer gaschromatograph equipped with a ilame ionization detector (hydrogen-air flame)and stream splitter.

Samples were trapped in 0.01 mm path-length AgCI infrared cells, usinga heated collection line (RIIK) and a Dry Ice ethyl alcohol trap.

Infrared spectra were run on a Beckman IRS.

r Fisher Chemical Co., Silver Spring, Md.

5

III. RESULTS

A. IDENTIFICATION OF ETHYLENE

The retention time of the gas produced by P. cyclopium wai identicalto that of commercial ethylene. The brominated derivative had a retentiontime identical to that of commercial 1, 2-dibromoethane and had an identicalinfrared spectrum.

B. RELATIONSHIP OF THE PRODUCTION OF ETHYLENE TO THE LIFF CYCLE

Penicillium cyclopium grows rapidly under the previously describedcultural conditions (Fig. 1), although the time required to reach maximummycelial mass varies with the amount of inoculum (Fig. 2). When the basalmedium was used, growth preceded ethylene production by more than 60 hours.Under all experimental conditions studied, ethylene was produced only afterthe growth had passed its maximum, as determined by dry weight. The cessationof growth was probably caused by lack of carbohydrate, as additional carbo-hydrate supplied at the time of inoculation resulted in greater dry weights(Table I). The loss in dry weight probably indicates the onset of autolysis.

Microscopic examination of the cultures verified that ethylene productionis initiated after the onset of sporulation. The appearance of the culturesalso changed from white to olive green prior to ethylene evolution.

Preliminary experiments indicated that the optimum pH of the mediumfor both growth and ethylene production is 4.8 at the time of inoculation.Figure 1 shows that the pH of the medium falls to about 3.6 during the logphase of growth and then gradually rises to about 5.9 during the initiationof ethylene production and probable onset of autolysis.

The respiratory quotient (RQ) was computed from the oxygen consumptionand carbon dioxide production for each 8-hour period. The RQ reaches amaximum of about 1.3 during the period of maximum growth and falls below1.0 soon after the culture begins to lose weight.

Figure 1 shows that no measurable ethylene (>0.05 ppm) is recordeduntil the growth rate has passed its maximum. This was true under allexperimental conditions studied.

C. RELATIONSHIP OF SUCROSE TO ETHYLENE PRODUCTION

Table 1 shows the effect of varying the sucrose concentration in thebasal medium on total dry weight and ethylene production. When the sucroseconcentrations were varied, the growth (mg dry wt) was proportional to thesucrose concentration, and total ethylene production was proportional togrowth. When the ethylene production is converted to microliters per gramdry weight, there is no significant difference among the four sucroseconcentrations tested.

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7

TABLE 1. EFFECT OF SUCROSE CONCENTRATION ON ETHYLENE PRODUCTIONS/

Sucrose, Dry Weight, Ethylene, Time,

girams/liter mg pliter/g dry wt hours

5 10 145 78-90

10 19 152 78-90

15 29 144 78-90

25 43 155 90-102

a. The sucrose concentration of the basal medium was varied.

Cultures were followed for 9 days and sampled for ethylene anddry weight every 12 hours. The reported values are the maximum

recorded during the 9 days. Time refers to maximum ethylene

production with maximum dry weight occurring prior to this.Each value represents the average of four replicates of threeflasks each.

D. RELATIONSHIP OF AMMONIUM NITRATE TO ETHYLENE PRODUCTION

Table 2 shows the effect of varying the ammonium nitrate concentrationin the basal medium en total dry weight and ethylene prodviction. Exceptfor the highest concentration, there is no significant difference in dryweights. Ethylene production was not proportional to growth. Maximumethylene production was obtaincd with 2 grams per liter and good ethyleneproduction from both 4 aud 8 grams per liter.

E. RELATIONSHIP OF NITROGEN SOURCE TO ETHYLENE PRODUCTION

Figure 3 shows the utilization of different nitrogen compounds byP. cyclopium over a 9-day period. The nitrogen compounds were substitutedsingly in the basal medium in amounts to give 1.4 grams of nitrogen perliter. Each nitrogen source tested was successfilly utilized by the fungusfor growth. Growth did not vary appreciably but the ethylene productionappears to be dependent on the nitrogen source, and not proportional togrowth. Ammonium nitrate, L-asparagine, and ammonium citrate yielded thegreatest amount of ethylene.

TABLF 2. EFFECT OF AMONIUM NITRATE CONCENTRATIONON ETHYLENE PRODUCTIONS/

NH4NO3 Dry Weight, 9thylene, Time,grams/liter mg pliter/g dry wt hours

1 47 22 114-126

2 50 172 90-102

4 48 146 90-102

8 45 124 90-102

16 36 21 90-102

a. The ammonium nitrate concentration of the basal medium wasvaried. Cultures were followed for 9 days and sampled forethylene and dry weight every 12 hours. The reported values arethe maximum recorded during the 9 days. Time refers to maximumethylene production with maximum dry weight occurring prior tothis. Each value represents the average of four replicates ofthree flasks each.

KNO3 ] \NH4NO3,o, DRY wVY 1120 170 1 \ 60

£0- - - -o. 1 o 40

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60 4120 120- "60

40 .480 a0.- " 40

20'410 40-. 20.

,L LAspragi,.

A , of40'- 50 17004 40

,o0 ,6 so 80r

20 i/ / .4

-40 40 0

N 44 6 a 10 2 A 6 a 10

DAYS GROWT"

FIGURE 3. Effect of Various Nitrogen Sources onGrowth and C214 Production. Each nitrogen source

was substittuted for the N114N0 3 In the basal mediumto yield a ntrogen concentration of 1. g/liter.

The cultures were grown at 25'2 C in thl, dark;different flasks wore sampled every 24 hours forC2114 arid dry weight. Each value represents theLaverage. of four replicates of three flasks each.

9

F. EFFECT OF ETHANOL ON ETHYLENE PRODUCTION

Since Hall,- Phan Chon Ton,' and Gibsoni, reported that feeding ethanolto P. digitatum resulted in a greater production of ethylene, this substratefor ethylene production by P. cyclopium was studied. Time-course studiesof growth and ethylene production by P. cyclopium grown in the basal medium,with and without ethanol, were made for a 9-day period (Fig. 4). Fourdifferent flasks were stoppered for each 24-hour period. Maximum U'ryweights for the various ethanol treatments were: Control, 52 mg; 0.1%,54 n; 0.5%, 51 mg; 1.0%, 50 mg; and 3.0%, 58 mg. Except for the highestethanol treatment, growth did not significantly increase with the adaitionof alcohol, but ethylene production did. In each treatment the ethyleneproduction occurred ifter maximum mycelial growth had been reached. Thegreatest amount of ethylene was produced when the mycelial mats began tolose weight. Ethylene production was stimulated, but was not proportionalto growth when ethanol was added to the basal medium.

G. EFFECT OF AN ALDEHYDE-FIXING AGENT ON ETHYLENE PRODUCTION

When ethanol was shown to have a stimulatory effect on ethyleneproduction, studies were initiated to inhibit normal ethanol production inthe fungus. Because no satisfactory method exists for trapping ethanol invivo, several aldehyde-fixing agents were tested in an attempt to blockthe conversion of acetaldehyde to ethanol. Extensive work by Neuberg inthe early 1900's as cited by Nord and Weiss.1 showed that sulfite andbisulfite salts were effective fixing agents of acetaldehyde. The useof other aldehyde-fixing agents, such as hydrazides, dimedone, andthiosemicarbazide, has the same effect as sulfite.'-

Our experiments with the sulfite and bisulfite salts showed them tohave an adverse effect on the growth of this fungus. Dimedone (5.5-dimethyl-l,3-cyclohexanedione) obtained from Eastman Kodak permitted goodgrowth with no observable growth differences from cultures grown on thebasal medlum minus dimedene. The dimedone was sterilized with a Seitzfilter and aseptically added to the medium before inoculation. Figure 5shows the effect of dimedone on total ethylene production. Dimedoneappears to bc a good inhibitor of ethylene production in this fungus.

10

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IiI

IV. DISCUSSION

The discovery that Penicillium cyclopium produced a gaseous metabolitechromatographically similar to ethylene prompted a more thorough char-acterizagion of the gas and the relationship between its production andthe growth curve of the fungus. Preliminary experiments indicated thatP. cyclopium grew well and produced ethylene on the basal medium and thatgrowth was more uniform on solid than on liquid media. Heating the solidimedium to liquefy the agar allowed determination of dry weights withoutdifficulty.

Studies with P. digitatum have shown that ethylene is produced through-out the life cycle. -e However, it appears from some of the publisheddata that although ethylene was produced continuously, the peak of ethyleneproduction did not occur until after the dry weight reached a maximum.10,1 3 .16

In contrast, P. cyclopium does not produce any ethylene (>0.05 ppm) untilthe dry weight reaches its maximum value. At the probable onset of autolysisthe ethylene production increases rapidly to a peak and within 48 hoursthe ethylene production declines rapidly to a much lower level, where itremains for the duration of senescence.

Althcugh a number of other fungi have been reported to produce ethylene,P. digitatum is the only fungus in which the structure of the gaseousemanation has been v5 rified by conversion to chemical derivatives. Con-sequently, we converted the ethylene produced by E. cyclopium to 1,2-dibromoethane and compared its infrared spectrum with that of authentic1,2-dibromoethane. The spcctra were identical, thereby proving con-clusively that P. cyclopium p~i.ces ethylene as a metabolic product.

Factors found to affect the growth of f. cyclopium did not alwaysaffect ethylene productipn the same way. When the sucrose concentrationwas varied, the ethylene produced was proportional to growth. Work withP. digitatum has shown that ethylene is closely associated with growth."

When the source of nitrogen was changed or the concentration ofammonium nitrate varied, ethylene production was not proportional to growthin P. cyclopium. It has also been shown that abundant growth does notnecessarily indicate high ethylene production in Penicillium digitatum.1

I Ok

Fergus'2 reports that P. digitatum does not require any specificnitrogen source to produce ethylene. If organic and inorganic nitrogencompounds allowed growth, ethylene was produced. Figure 3 shows thatP. cyclopium when grcwn on various nitrogen compounds, always producedethylene but the amount was dependent on the nitrogen source.

Ethylene production is shown to be affected not only by sucrose con-centrations but possibly more significantly by nitrogen metabolism. Sinceethylene production does not begin until the onset of autolysis in thisfungus, it appears likely that the role of nitrogen is that of proteinnitrogen during senescence. The respiratory quotiont during ethyleneproduction could reflect the metabolism of some proteins as well as fats.

12

Ethanol was found to stimulate ethylene production of P. digitatum bya number of workers .- e Only a few of the mny comp mids tested have shownsuch a marked stimulation of ethylene In the Penicilla. Ethanol is anappealing precursor of ethylene.

Penicillia are ethanol producers and acetaldehydc nky be found eitheras a precursor of ethanol or during its oxidation,)" Numerous other fungihave also been shown to produce ethanol both aerobically and anaerobically. 1 ' '

Many of the fungi reported to produce ethylene have also been shown to begood alcohol producers. Among the highest ethanol producers is Asperg]illusclavatus, ' l which, interestingly, is reported to be the largest ethyleneproducer of the fungi examined by Ilag and Curtis. '

Because a specific inhibitor of ethanol synthesis is not available, analdehyde-fixing agent was used. An experiment was carried out to fix theacetaldehyde, a primary precursor of ethanol in yeast fermentation, withdimedone. As an inhibitor dimedone does not appear to have any visibleeffects on the growth of the fungus, including the dry weight. As shown inFigure 5, dimedone, at a concentration of l0- M, inhibited 70% of the totalethylene production. Both ethanol (Fig. 4) and acetaldehyde (Fig. 5) havesignificant effects on ethylene production. Acetaldehyde, unlike ethanol,is a very poor precursor of ethylene.i"°11C

Figure 2, on the effect of inoculum size on growth and ethylene, showsa relationship similar to one observed when Penicillium notatum is grownfor penicillin production. In general, conditions that favored the fastestgrowth of the fungus yielded the most penicillin. This requirement forfast and abundant growth also appears to yield the most ethylene in P.cyclopium. This may help to explain why (Table 2) thc lowest and highestconcentrations of ammonium nitrate yield significantly less ethylene, whenthe other concentrations have been shown to produce large quantities. Thisexperiment indicates that inoculum size should not be over looked whenconsidering variability in ethylene production from fungi.

13

LITERATURE CITED

1. Young, R.E.; Pratt, H.K.; Biale, J.B. 1951. Identification ofethylene as a volatile product of the funguis Penicillium digitatum.Plant Physiol. 26:304-310.

2. Dimond, A.E.; Waggoner, P.E. 1953. The cause of spinastic symptomsin Fusarium wilt of tomatoes. Phytopathology 43:663-b69.

3. Ilag, L.; Curtis, R .W. 1968. Production of ethylene by fungi.Science 159:1357-1358.

4. Nickerton, W.J. 1948. Ethylene as a metabolic product of thepathogenic fungus, Blastomycers dermatitidis. Arch. Bioichem.17:225-238.

5. Fergus, C.L. 1952. The nutrition of Penicillium digitatum Sacc.Mycologia 44:183-199.

6. Abeles, F.B.; Rubinstein, B. 1964. Regulation of ethyleneevolution and leaf abscission by auxin. Plant Physiol. 39:963-969.

7. Burchfield, H.P.; Storrs, E.E. 1962. Biochemical applications ofgas ehromatography. Academic Press, New York, N.Y.

8. Hall, W .C. 1951. Studies or. the origin of ethylene from planttissues. Bot. Gaz. 1l3:55-u.

9. Phan Chon Ton, M. 1960. Nouvelles observations sur les substancescapables do stimuler la formation d'ethylene par le Penicilliumdigitatum. Compt. Rend. 251:122-127.

10. Gibson, M. S. 1963. The biogenesis of ethylene. Ph.D. Thesis, PurdueUniversity, Lafayette, Indiana.

11. Nord, F.F.; Weiss, S. 1958. Fermentation and respiration, p. 323-368.In A. H. Cook (ed.) The homistry and biology ef yeasts. AcademicPress, New York, N.Y.

12. Rose, A. H. 1961. Industrial microtbiology. Butterworth and c.Ltd., London.

13. Fergus, C.L. 1954. The production of ethylenie by Penicilliumdigitattum. Mycologia 46:543-555.

14. Meheriuk, M.; Spencer, M. 19b4. Ethylene productioi duringgermination of oat seeds and Penicillium digitatum spores. Can. J.Bot. 42:337-340.

15. Phan Chon Ton, M. 19)57. Observations stir la production d'ethylenepar le Penicillium digitatum Sacc. Compt. Rend. 244:1243-1246.

14

16. Spalding, D.H.; Liederman, M. 1965. Factors affecting the productionof ethylene by Penicilli'Am digitatu . Plant Physiol. 40:645-648.

17. Cochrane, V.W. 1958. Physiology of fungi. John Wiley & Sons, Inc.,New York, N.Y.

18. Foster, J.W. 1949. Chemical activities of fungi. Academic Press,New York, N.Y.

II

17

UnclassifiedSecurilv Classificationi

DOCUMAENT CONTROL DATA. R & D(50twity eloairldrtie, of t#$no, bedf' of abstract and kideettd afftnolaffe must b. enltni when, the .veguIlfRvn I@ cleseellgfd

1 . ORIGm IN eAcTI ViTV fCoopmtie "to. So. RE*ORT SECURITY CLASSIFICATION

PRODUCTION OF ETHYLENE BY PENICILLIUN CYCLOPIUM

a. DESCRIPTIVE NOTES (Tjr0 o elept and fact" Ne dets)

0. AU TNORISI1 (Pi.t Rims. ldddl M181aUl, fact e"P)

Joseph (NMI)'LonskiHarry E. Cahagan III

0. REPORT OATS 70. TOTAL NO. OP rPAGES 17b. NO. OF 0190S

April 1969 17 18Se. CONTRACT OR GRANT NO. S.ORIGINATOMWI REPORT MNMMIER45t

6. PROJECT NO. 1B5626O2AO61 Technical Manuscript 532

C. SOb. OTNER REPORT NOMS (RAeth 4101RMee" OtSW be 011018"dtift. roe"p)

10. OISTRIGUTION STATEMENT

Qualified requesters nuiy obtain copies of this publication from DDC.Foreign announcement and dissemination of this publication by DDC is not authorized.Release or announcement to the public is not authorized.St. SUPPLEMENTARY NOTES 12. SPONSORIMG MILITARY ACTIVITY

Department of the ArmyFort Detrick, Frederick. Maryland, 21701

IS. AMIYRACT

Ethylene production by Penicillium cyclopiu!n, ATCC No. 7615, was studied inrelation to the life cycle of the funguts. Ethylene, --..5ppm, was not produceduintil after the cul1ture reached its maximum dry weight. When the sucrose con-contration of the ifediun was varied, ethylene was proportional to growth. However,whien the armnontuin nitrate concentration was varied or the nitrogen source changed,there was no direct relationship. InOCL1lUM size a iSO influenced ethyleneproducti on. Ethanol markedly stimula ted total ethylene product ion; dimedone wasa good inhibitor of ethylene production.

The strtlcLure of the naturally produced ethylene was verified by convertingit to 1. 2-dibromoethane and showing that its infrared spectrum was Identicaltu that of authentic 1. 2-dibromoethane.

14. Key Words*Ethiylene*PeniCihliiim*SLucrosvI

EthtaoolsInhibitorsProduict ionFungi

NitrogenLife cycles

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