Spore Germination and Carbon Metabolism in Fusarium solani.III. Carbohydrate Respiration in Relation to Germination 1,2

Vincent W. Cochrane, Spencer J. Berry, Frank G. Simon, Jean Conn Cochrane,Calden B. Collins, Jay A. Levy, and Peter K. HolmesDepartment of Biology, Wesleyan University, Middletown, Connecticut

In earlier papers of this series (9, 12), it has beenshown that macroconidia of Fusarium solani f. phase-oli require exogenous carbon for germination, and thatendogenous respiration contributes to but is quantita-tively insufficient for the germination process.Only certain sugars can support germination; in thepresent paper we have first examined the respiratoryresponses of spores to utilizable and nonutilizablesugars, and determined the major features of theoxidative assimilation of glucose.

There is some evidence from work with bacteriathat growing cells respire glucose by a different bal-ance of pathways than do resting cells, in particularthat respiration during growth involves a larger par-ticipation of some pathway preferentially liberatingcarbon atonm 1 of glucose than does the respirationof nongrowing cells ( 1, 2, 31 ). Chlamydospores ofTilletia caries clearly differ in their pattern of oxida-tion of labeled sugars from mycelium (29). As anapproach to this problem as it relates to spore germi-nation, we have compared the metabolism of un-germinated spores with that of germinated spores,and the responses of ungerminated spores to exogen-ous amiinonium nitrogen. The purpose is not to es-tablish a particular and specifiable numerical balanceof pathways, but rather to use the observed changesin respiratory pattern, if any, as a first indicationof the events which are characteristic of or crucialto spore germination and growth. A spore of thistype appears to be a useful system for the analysis ofmietabolic differences between growing and restingcells, since growth requires specific exogenous nu-trients and is marked morphologically by swelling ofthe spore and germ tube formation.

Macroconidia of F. solani f. phaseoli require oxygenfor germination; at the same time there is good evi-dence in other Fusarium spp. for an active anaerobicrespiration of sugars by resting mycelial cells (10,16, 17. 19, 33). Examination of fermentative capa-city in macroconidia of F. solani f. phaseoli shows that

1 Received Feb. 1, 1963.- This investigation was initiated with the support of

granit-ini-aid (MET-18) from the American CancerSociety on recommendation of the Committee on Growthof the National Research Council, and has been completedwith support from the United States Atomic EnergyCommission under Contract AT(30-1)-1813, and fromthe National Science Foundation under Research GrantG-9087.

the ungerminated spore is unable to consume glucoseanaerobically; a weak fermentative capacity is ac-quired during germination.

Materials and MethodsThe strain of Futsariiumii solani f. phaseoli was ob-

tained from Dr. XV. C. Snyder; it is pathogenic tobean, and under our cultural conditions producesonly macrospores. Spores were harvested from aglucose-aspartic acid-yeast extract medium (9), andwashed twice by centrifugation and once by filtra-tion. Spores were germinated in a stirred and aerat-ed jar for 12 hours at 250; the complete system con-tained: spores 2 g per liter (wet weight), Difcoyeast extract 0.5 g per liter, potassium phosphatebuffer (pH 6.5) 0.012 i\r, (NH4)9S04 0.002 M, andAMgCl9 0.002 .i.

Lipid was determined by 24-hour extraction withpetroleum ether (Skelly Solve B) in a Soxhlet ap-paratus. Carbohydrate was estimated by the an-throne method (28), mannitol by a periodate method(38). Respiration was measured at 300 by conven-tional methods (36); endogenous values are subtract-ed from all readings for substrate oxidation. Unlessotherwise stated, \Varburg flasks contained potas-sium phosphate buffer (pH 6.5) 20 umoles, MgCl.,10 AL moles, spores and substrate as indicated, alkaliin a center well, and a total fluid volume of 3.2 ml.Spore dry weight was determined from an aliquot ofthe suspension used.

The sequential formation of C1402 from labeledsubstrate was determined in an apparatus consistingof two 125 ml Erlenneyer flasks fitted with standardtaper joints (24/40) at the top and with male andfemale ball joints (35, 25) 1 cm above the base, pro-jecting laterally. Two such flasks were held to-gether by a ball joint clamp; one contained the me-tabolizing system. the other standardized Ba(OH) .The flasks were capped and shaken in a water bathat 30°; at intervals the alkali flask was replaced.All operations with open flasks were carried out ina stream of C02-free air. The absorbed CO wasdetermined by titration, and carrier Na2CO3 wasadded if necessary; the precipitate was then filteredon a sintered glass planchet and counted as an in-finitely thick layer in a gas-flow windowless propor-tional counting system. Radioactivity in cells and infractions extracted froml cells was determined by

533

Table IRespiration of Stl(oars

\W ashed spores w ere inlcubate(d 4.5 lhr w ith 10 ,unioles of glucose, fructose, or Ilail)iCs OIr ; Illoles ot tirlialose.Thle dry weight of spores per flask \xas 13.8 mg ( ungerminated spores ) ancd 14.1 mig (gerininate(l spores ). (xeriii-nationi of si)()res anid otlher constituents of tlle respiration vessels have heen lescrilied in Materials and(I Metihods.-\1l values xx ere cirreeteil toren(ha(:)gTeefn(u, resj)iratiponn 1v nsubttraction of the 0) uptake in (ontr flasks lackingsubstrate.

tn ,erninated spores

7.102.682.54

0.,up)take,(, *

16.78.58.4

18.8

(icrinlinated( spior('sSug-artilizedl,

4.ti( des10.09.79.84.9

8.2.92

2.49i,7.1.)

l peir lhr per lng (lry spores. () to 12(0) iiiuinntes.As per cenit of tile C), uptake calcnlated for (o(mpl)lete aerohic resp)iratiion (f the snioar di sappearin, int

uni in 4.5 lhours.

coulnltingo ilnfillitel, thlill la ers iII the s'ame S StemllthilS llethio(d wxas cdlibrated lIx coliluhstiol of t)-Iiseof known activitx

(1iicose-34-jari'till ( ilhlis; otilh.i laheled emo llpotill(is w ire eonll-

XlI sv\teills folr lap'r ehronall to -rapy)h \xer-e (lrawixxfir)l thle i1ailtial (ot Smith 34

Results

tiliso/ioll of S"own's. MIlla iSCe 0(ld rlruetose (ii

lint Sul)l)pi't poM'e gerilli t1ati i)1, g tio'sC(.and tr'elhhlose.'

lo ( T'lie (hta of tbldde I slhox\ that the 2 first-

lialuleu stig ars are ox idatdli 1\\ 1 thaill iticose

,)I- trelold.SC, lld esplec'i ally\ that g-'111 n11ation re'sults

ill I() alccel erati of thle i'at ot o\idatioii of thle lnoll-

utili]Iliale nti--,'ti

All 4 stgai'rs (i ppeare m the nlle(li tllm the

( ltath thelrCfor'e st1igge>St 0I ididti VC LX1.1111 ti011 ot Is

iiit'1h (is (02 of the lulga' s"upplied. Strictly speak-

illg-. (O.ix idtit V (e s 11i ilati n11 1h .s heCen i 0111o\ Wxithl

g-lue iet'. iillce ( n)II\ \xwith it as5 Isub strate has thle mde(li-lim heell piwox ed to cmOntailn 110 011dbl e pnri(ticts thi

W aLs (lete rini ne(il Vy pro i lle( C' s stu -

stl-a[te. follo)\Vedxl l)\ den 0lo stl'Ltioi tllat 1e.SS tlalotf the glucis)se lahel i'cmaiaii iii the iiiemliiu iii. FI0\\

c vie', it seeniI uni ilelv that the iIIetallhO iISI Im Ofirtui tose .0' malnan )se otild ix\ I'ir t to 0o )hl pro)(1-

icts in it foi' eile(I frtg0 lue

'lhe r'atiO otf s1-aro' i(xldized to sgioar' assmiilatemi

iX ulpp'Oinxllatelv (0 2 foi' g-UtiOXse anl tirehal ose. ().08

to 0.09) foi' flruictose and mlannise. Thus, the (Xilda-

ti i)nfof ne miole of maninose m1 fr'uctOise tust, unless

enog-enotus ieslpiiatiOllci01iltributes. supply eneigy

fiO1' the assin1ilattio of 11 12 moles although this

valuie seems hih-11, it is n1ot ilmissihly so ii1 the as-

1uii tio that ass in ilaftio su i'nriolectule i'e-

(Iuires the hi'eakd(oxxn oIf iiiilx oni niolec tle iif A l'and1(1 tle futi'tlhei' alssip11p1)tioln that cmi npdete . i(a-

tiiii o0f one molc of hlexose xielils 38 miole> oI XA tP.

Trehali )se titilizatih i s ll( w\\ llat .sloxxvci thl;lllthalt ot g)-luc).se 111(1 less ) e to grerrn na ti(itthils pi()hahly mleanls that h\r(ilniis otr the (i saceiho-i(le iS tiie iate-li iuitil, tepl). (ialato.tse is iiot r-C'pir-etl lx tlilgl- lil te (lpe( or-e>S \\ tit nill 3 honrS. n( wil\slo(-l(\I ) 1.1 lv -erillillated spot -c. I In1 -

25

20?-

15

10

- QOo(E) Qo2(G) YIELD

5

1 2 4 1 2 4 1 2 4

RELATIVE SPORE LOADFic,. l. Thll l-]atiO, ,t si,oi-c lolad pcrl tlabsk t c ll-

erocoi nidia. Actual s1(rc ()aills 275, 1dI1.1) in- lir

eig-ht per W\'xarhur, flask, tlimle 5 11umrs. o, Fi-ate ()f einl(()g-nl(i S iresp0iratioii ill Al pc- ih ir- t r I

pi res ( i(() -ate ()t irespirati(iii xxithi ol n-I se (

pin lIes lwter fiask ) (corr-et ed( f cmild(wluimins r cspt ir tit

inI yl per lioiir ptr Img sesP ri ( )., ii]pthlk 'is ip r

cenit o2- that re(qiire(d C()i- c(mipl ete ()\i(h(ti(at ti.HIt('s)' utilize-.d.

Stig-ar

GlucoseFruct se

MIanrnoseTrehalose

>ingarntili,'ed,

9.89.6

4.8

nlptakc.( *

26.3,r. .(

9.0:7.8

2_ 0

534 PLAN>-T PHYSItOLOGY

COCHRANE ET AL.-METABOLISM IN FUSARIUM

Table IIThe Effect of 02 on Respiration of Ungerminated and of Germintated Spores

Q02_(E) **Q_Qo2 (G)***Spores Density* Air 02 Air 09

Ungerminated 9.5 4.4 5.0 4.4 5.219.0 4.21 4.16 2.3 2.5

Germinated 5.9 22.7 21.3 15.2 16.111.8 20.7 24.017.7 19.5 23.3 4.90 4.66

As mg dry weight per Warburg flask.02 uptake, jul per hr per mg dry weight of spores in absence of substrate, 0 to 120 min.02. uptake, 1Al per hr per mg dry weight of spores in presence of glucose (20 ,Amoles per flask), corrected forendogenous respiration, 0 to 120 minutes.

nitol is oxidized rapidly by both, and supports normalspore germination.

It was of some interest to determine whether theextent of oxidative assimilation is dependent solely onthe amount of substrate provided or whether the as-similation is greater if more spores are present.The data of figure 1 indicate that the extent of oxida-tion of glucose, expressed as a fraction (per cent) ofthe theoretical oxygen uptake for complete combus-tion, is practically invariant with spore load. Thatis, the extent of oxidative assimilation (measuredby the missing oxygen uptake) is dependent on thesugar provided and not on the number of spores.Dawes and Holms (13) report the same phenome-non in Sarcina lutea.

The uniformity of the extent of glucose oxidationis another argument for the absence of an effect ofglucose on endogenous respiration, already suggest-ed by other data (12). The oxygen uptakes fromwhich yield data in figure 1 were calculated were firstcorrected by subtracting endogenous values, deter-mined in control flasks lacking substrate. If sub-strate suppressed endogenous respiration by somefraction, the corrected oxygen uptake with glucosewould be affected differently at different spore den-sities and the yield would not be, as it is, invariantwith density.

These data refer to germinated spores; ungermi-nated spores respond similarly over the range tested,3.4 to 13.5 mg (dry wt) per 3 ml, although the lowerQo., of ungerminated spores results in rather lessuniform data.

It was somewhat surprising to observe, as shownin figure 1, that the Qo., with glucose (corrected forendogenous respiration) is inversely proportional tospore density, although the endogenous Qo., is es-

sentially invariant with density. This was true atany time period measured within the total time of theexperiment, and was as true of ungerminated sporesas of germinated.

These data suggested that glucose respirationmay be more sensitive to oxygen tension (and henceto spore density) than is endogenous respiration.This possibility is rendered unlikely by the data oftable II. Some effect of increased oxygen can be

seen, but it is not consistent and not of the magnitudlenecessary to explain the density effect shown infigure 1.

Since alkali was present in all flasks and the sys-tem was buffered at pH 6.5, it is unlikely that CO,accumulation is responsible for the density effect.

When the spore: glucose ratio is varied by in-creasing the glucose provided at a fixed spore den-sity, there is no effect, over the range 5 to 20 /Amolesglucose per 3 ml, on the Qo2; as shown earlier (12),there is a linear relation between total O., uptake(corrected) and glucose supplied but no rate re-sponses can be detected.

For the present, the differential effect of spore

density on endogenous and substrate respiration mustremain unexplained. From the aspect of method,however, it is clear that comparative studies of therespiratory rate must, at least with this organism, becarried out at a fixed spore density.

Products of Oxidative Assimtilation. The highlevel of oxidative assimilation, coupled with theearlier finding (9) that oxidative assimilation canlsupply some of the carbon requirement for germinia-tion, suggested an analysis of the metabolic fate ofassimilated glucose. In particular, it was of interestto determine whether carbohydrate is converted tolipid. We have shown earlier (12) that growth athigh glucose concentration increases the lipid coni-

tent of spores; Sisler and Cox (33) observed inFusariumn roseumnz conidia incubated in glucose an ac-

cumulation of granules reacting with a fat stain.Other experiments have shown that cold 5 % tri-

chloracetic acid or boiling distilled water are as ef-fective as boiling 80 % ethanol in the removal of cellactivity.

The data of table III show, first, that about 75 %of the activity of the glucose used appears in thecells; most of the remainder is recovered as CO,and the soluble products (supernatant) contain only5 % of the original carbon-14 supplied.

In other experiments, we have been unable to de-tect the formation of acetaldehyde by prolongedl as-

piration of air through a metabolizing resting-cellpreparation and then through a solution of 2,4-dini-trophenylhydrazine. A volatile acid, presumably

**

535

Table IIIMajor Products of Glutcose Assimnilationi by Ungermninated Spores

Spores were shaken 5 hr at 300 in flasks containing: spores 145 mg (dry wt), glucose-U-C14 150 ,umoles (2.28JAc), potassium phosphate buffer (pH 6.8) 60 unmoles, MgCl, 30 MAmoles, and water to 10 ml. The connecting flask(see Materials and Methods) contained 10 ml of 0.4 \ NaOH. Data are from 3 pooled flasks, but only 334.1 mg ofdried cells were taken for extraction.

The dried cells were extracted for 16 hr with petroleum ether, anid the extracted lipids were dried, weighed, andcounted. The residue was extracted 3 times in boiling 80 % ethanol. The residue from the ethanol extractionwas heated at 1000 for 3 hr in 20 % KOH. An aliquot of the KOH-extracted material was removed for counting,and to the remainder was added 4 volumes of 95 % ethanol. The precipitate was washed with 100 % ethanol, dried,weighed, anld counted. The final residue represents material inisoluble in petroleum ether, 80 %, ethaniol, and 20 %KOH.

Material anialyzed

Glucose utilizedNMetabolic CO.,SupernatanltCells

Lipid fractionEthanol extractKOH extractEthaniol precipitateResidue

Recovery from cells, %c

DryWeight,mg

27.0

334.137.093.480.97.9

97.892.7

TotalActivity

m,uc2280417109

175058

118022461118

SpecificActivitym,uc mg

84.6

5.251.57

12.62.777.721.21

TotalActivity,

1003.3

67.412.83.56.7

90.2

* Cell total activity takeni as 100 (.

acetic, is formed by resting cells acting on glucose.but only in very low yield; in a typical experimentthe oxidation of 3 mmoles of glucose yielded 106 ,umoles of volatile acid.

Amino acids occur in the ethanolic extract ofcells, but have not been studie(d in any detail; asparticacid, alaninie, serine, glutamiiic acid, and valine havebeen i(lentified by paper chromatography and shownto become labeled during incubation with uniformlylabeled glucose.

It appears from tllese (lata that glucose does notenter appreciably into lipidls during oxidative as-

similation. The bulk of the activity is in the ethanolextract; it slhould be noted, however, that the specificactivity of the polysaccharide fraction is second onlyto that of the ethanol-extractable fraction. This sug-gests a considerable exchange between the polysac-charide and exogenous glucose via some componentof the etlhaniol-soluble fraction.

The small incorporation into lipids and the exten-sive incorporation into an ethanol-extractible frac-tion are quite similar to the results of de Fiebre andKnight (15) with myceliumii of Penicilliiu;;i chlr-sogenuml.

In order to determine mlore nearly the compoundsinto which glucose carbon enters, the hot water ex-tract of cells incubated with labeled glucose was frac-tionated by the method of \Vhistler and Durso (39).All fractions were analyzed for total radioactivity.carbohydrate, and mannitol. Results appear in fig-ure 2.

The major component eluted with water wsas iden-tified as mannitol by paper chromatography in the

follow-ing solvents: ui-butanol-acetic acicl-water (4:1: 5). isopropanol-water (4: 1). an(l ethyl acetate-pyridline-water (60: 25: 20), following procedures(lescribed by Smith (34). AIannitol was; visualizedlby a silver nitrate dlip. The identity of miainnitol wasverified by co-chromatography with authentic man-nitol-1,6-C14, the strips being scanned Nith a NuclearChicago Actigraph system:; with all solvents the ma-jor C'4 peak coincided with the reference compound.

The first carbohydrate peak shown in figure 2 isan artefact-mannitol at high concenitrationi reacts

K - WATER >I*-ETOH,5%K--ETOH,IO%----H

0

0.K

I-U4c-j49I~-0

hi

i

I0-iI2

FRACTION NUMBERFIG. 2. Fractionation oIn a Celite-charcoal column,

200 X 25 mm, of the hot water extract from 219 mgmacroconidia incubated 4 hour at 300 with 450 ,umolesglucose-U-C14 (3.66 ,uc). A=total activity, C=car-bohydrate (as glucose, by anthroine method), M=nman-nitol. Fraction volume 100 ml, eluting solvents indicatedltop of graph.

536 PLA-N'T PHYSIOLOGY

COCHRANE ET AL.-METABOLISM IN FUSARIUM

I.-ai0

hi

hi

-J3

537

L2 4 6 0 2 4 6 Z 4 60 2 4 6TIME, HOURS TIME, HOURS

FIG. 3 (left). The effect of germination on the cumulative radiochemical yield of CO2 from specifically labeled glu-cose. Ungerminated (U) and germinated (G) spores, 100 mg dry weight, incubated at 300 in medium containing glucose150 Amoles, potassium phosphate buffer (pH 6.5) 60 ,umoles, and MgCl, 30 umoles, volume 10 ml. Glucose-3,4-C'4provided at a total activity of 100 m,uc, other sugars (glucose-1, -2, -6-C"4) at 2.0 ,c. Curves numbered to ilndicatethe sugar used; the arrow marks exhaustion of the glucose.

FIG. 4 (right). The effect of exogenous nitrogen, supplied as (NH4)2HPO4 at 40 ,tmoles, on the cumulativeradiochemical yield of CO2. Spores, 93 mg dry weight, incubated with glucose-3,4-C"4 at 80 m,uc with other sugars(glucose-i- and -6-C14) at 2.0 ,uc, in medium containing glucose 150 Amoles, potassium phosphate buffer (pH 6.5)60 umoles, and MgCl2 30 gmoles, volume 10 ml. Curves numbered to indicate the sugar used; arrow marks exhaus-tion of glucose.

(pink color) with the anthrone reagent. The an-throne peak of fractions 7 and 8 is presumably, inview of its elution with 5 % ethanol, a disaccharide;paper chromatography of these fractions showed a

compound with the RF of sucrose in ethyl acetate-pyridine-water, but the specific p-anisidine reagentdeveloped no color. Hence it is not possible to spe-cify the carbohydrate of these fractions. Nor hasthe material reacting as mannitol in fraction 1 beenidentified.

The most striking feature of oxidative assimila-tion is the contrast between it and the formation ofreserves during growth. Spores as harvested froma glucose medium have a negligible mannitol and ahigh lipid content (12), while assimilated glucosehardly enters the lipid fraction and is instead foundin considerable part as mannitol (fig 2). Mannitolis so easily washed from the spore that it would ap-

pear to be inadequate as a permanent storage prod-uct; in Fucus vesiczulosus it has been shown (4) thatmannitol is a temporary depot only.

Oxidation of Specifically Labeled Glucose. Apreliminary communication from this laboratory(11) noted that spore germination in F. solani f.phaseoli is accompanied by a decline in the ratio oflabeled CO. from glucose-l-C'4 to that from glucose-6-C14. Work elsewhere, especially that of Cheldelinand coworkers (8) has shown since that if any con-clusions can be drawn from CO, data a greater range

of sugars must be tested. Katz and Wood (25) dis-cuss the ambiguities of all CO, methods.

In figure 3 are presented the cumulative radio-chemical yields of C1402 from specifically labeledglucose, defined as the ratio of total C14 in CO. to

the total activity of the glucose utilized. In figure 3ungerminated and germinated spores are compared;for both spore stages, CO2 formation from carbons3 and 4 is more rapid than from other positions.Germination is accompanied by increases in CO, out-put from all sugars studied, but the greatest propor-tional increase is from glucose-6-C'4. Output fromthis sugar is increased by a factor of 6, that fromother sugars by factors of about 2.5.

It is of some theoretical importance whether thischange in metabolism with germination represenltssome complex adaptive system developing slowlyduring germination or whether it can be more closelydefined in terms of known metabolic reactions. Con-sequently the simpler system shown in figure 4 was

tested; here are compared the respiration of glucoseby ungerminated spores in the presence and absenceof ammonium nitrogen. In general, the effect ofexogenous nitrogen is similar to the effect of germi-nation; in particular, although all sugars are respir-ed more rapidly and more nearly completely, therelative effect is greatest with glucose-6-C'4. Glu-cose-2-C'4 was not included; in other experimentsoutput from this sugar parallels that from glucose-i-C14 at a slightly lower level.

Since the ratio of C140, output from glucose-i-C14 to that from glucose-6-C14 is so often used as anapproximate index of respiratory pathways, it is in-structive to look at the time course of the CJ/C6 ratio,shown in figure 5. These values are drawn fromthe experiment reported in figure 4. Both with andwithout nitrogen the ratio declines steadily with time,and no one value can be considered characteristic.Nevertheless, the 2 curves shown are parallel, i.e.,

z

la-IL-Jhi

hi

0

PLANT PHYSIOLOGY

if).

0I_

hi

0

4c

4F

1 2 3 4 5

TIME, HOURSF1;. 5. The effect of exogenous nitrogeni oIn the

ratio of radio-chemical yields in CO. from glucose-i- and-6-C'4. Data from experiment shown in figure 4. -N= coiitrols without nitrogeI,-+N = solutioins with amil-

ioniiUini iiitrogen.

the (lifferential effect of exogenous niitrogen is (le-tectable at all times.

Both gernmination an(d the provisionl of nitrogenl)ring about a striking decrease in the assimiilationiof carbonis 3 and 4 of glucose into the cell; the con-

version of these carbons to CO., rises from about 30to about 70 cy. These (lata suggest acceleration ofanl Enmbden-Meyerhof system, in w%hich these carbonatoOn1s are convertedl to CO. by the decarboxvlationotf )yruvate.

It shoul(d he recalle(d that the provision of amii-

loliumii nitrogen andl glucose is necessary but notsufficienit for spore gernmination; sonme other factor.present in yeast extract andl replacedl by ethaniol or

acetoin. is re(Juired (9). The sinmilarities of figures3 ani(l 4 suggest that the governinig factor in theresp)iratory response to germination is the avail-abilitv of ammloniunm nitrogen and, presumably, theoccurrenice of synthetic rea'ctions which are (lependentoni a supply of utilizable nitrogen, and affect the bal-ance of (lifferent catabolic respiratory reactions.These conisidlerations are discusse(d later.

The mletlho(d of Heath and Koffler (18) was alsoal)plie(l to this nmaterial, although its sensitivity isleast for systenms producing CO, nmore from carbons3 an(d 4 of glucose than from other carbons. Germi-nate(l spores and ungernminated spores provided withammiiiiomiiunm nitrogen produce C14O. from glucose-l-

-U-C14 in amlounts suggesting practically no par-

ticipation of a pathway involving preferential libera-tionI of the carbon-1- of glucose as CO . Ungermi-lated spores without exogenous nitrogen, on theother hlandl, yield values ranging froml 4.6 to 15.2 %as the nminimum participation of such a pathway.These experimlents agree with those reported above

(fig 3-5) in that either germination or nitrogen ap-pears to shift imietabolisnm toward a nmore nearly equalconversion of carbons 1 and 6 of glucose to CO,.

Data so far presented have included only thelabeling of CO,. In table IV are data from an ex-perinment in which the relative contributions ofcarbons 1 an(d 6 to pyruvate were measured. Sincethe spores (lo not accumulate appreciable pyruvate,a carrier nmethodl was use(l, based of course on the as-sumption that metabolic pyruvate mixes with exog-enous pyruvate. Spores do oxi(lize pyruvate, al-though rather slovly, but to what (legree isotopicequilibriunm is in fact reached is unknown. For ourlinmited purpose, the fin(ling of a Cj1C6 ratio inpyruvate of 0.71 is an indication that the differentiallabeling of CO. (fig 3-5) is reflected in the dlistribu-tion of glucose carbons at the pvruvate level.

Table IVInicorporationi of G,lucose Carbonis into Pyruitatc

Ungerminatedl spores were incubated 3 hr at 300 induplicate flasks containiing: potassium phosphate buffer(pH 6.5 ) 60 umoles, glucose 100 Anmoles, unlabeled Napyruvate 200 /nmoles, MgCl., 30 ,umoles, and water to 10ml. Duplicate supernatants were pooled; pyruvate acidwas isolated as the 2,4-dinitrol)lheniylhydrazone, re-crystallized, and counted.

C14 in pyruvic acidSubstrate

Glucose-i-C'4, 8.68 mnAcGlucose-6-Cl4, 7.72 miAc

m,uc

51263.>

Yield, % *

5.98.3

C' 4 in pyruvate as per cent of that in glucose utilized.

A.naerobic Respiratiton. As mlentione(l earlier,the ability to ferment sugars is commnnoin in the genus

Fusarium, andl there is good evidence that theEmbden-Meyerhof pathway is operative. Ungermii-nated washe(d spores of F. solanii f. phaseoli (lo not.however, forn (letectable amounts of CO., anaerobic-ally from glucose (table V). Germlinated spores are

capable of a slow fermentation; the highest rate we

have mleasuredl is 7.8 ,ul CO., per hour per nmg of(lry weight. In the experiment shown in table V,an(l in others, the manometric anid( radliochemical(lata approximlately agree aind indlicate that about10 %c of the total glucose carboin utilized is convertedto CO2. If we assunme an Emndlein-Meyerhof path-way the conversion shoul(d be one-third; this suggestseither that fermentative assimilation occurs, or thatin this species anaerobic respiration proceeds bysome other mechanisml.

Ethanol is produced in the fermentation of glucosein approximately the sanme nmolar amount as CO*.as determined by an enzymnic methodl (5). Mycelialcells ferment but even mlore slowly than germinatedlspores; 72-hour cells froml shake culture fornm CO.at rates no higher thani 2.5 IAl per hour per mlg of dryweight.

-J4I--0

w

4c-Jw

L00

4

I I I IAs I I

538

I 0

OF6F

COCHRANE ET AL.-METABOLISM IN FUSARIUM

Table VAntaerobic Respiration of Germinated and

Ungerminated SporesUngerminated spores (8.7 mg dry wt) were incubated

with 28.7 m,mc of glucose-U-C14, germinated spores (7.5mg) with 83.5 muc glucose-U-C14. Substrate flaskscontained glucose 25 /imoles, 0.2 ml 2N H,SO4 (in side-arm, tipped at 4 hr), potassium phosphate buffer (pH6.5) 20,umoles, MgCl2 10 ,umoles, and water to 3.2 ml.Flasks were gassed with prepurified nitrogen; all valueswere corrected for dissolved CO, in reagents. Mano-metric and radiochemical yields are based on the glucosedisappearing; residual glucose was determined with theanthrone reagent.

Ungermi- Germi-nated natedSpores Spores

Glucose used, ,umoles 0.1" "9m,MAc 0.1

Endogenous CO.,, Al 3Substrate CO.,, ,ul 1C'4 inl CO.,, nmAc 0.02CO., yield, manometric, ...

CO., yield, radiochemical % ...

14.046.820218

4.4511.59.5

Ungernminated spores were incubated under nitro-gen-carbon dioxide (95: 5) in bicarbonate (36) ; no

detectable acid was formed by 9.5 mg (dry wt) ofspores acting for 3 hours on glucose.

The rate of anaerobic CO., formation from glucosewas not affecte(d by ergosterol and Tween-80 at theconcentrations used by Lindenmayer (26).

The data of table VI were obtained in an effortto determine whether the capacity to ferment isgained before germination is complete and whetherin fact it cannot be gained under conditions whichaccelerate metabolism without allowing germination.Spores were preincubated for 3 hours in contact withcombinations of glucose, ethanol, and ammoniumion: these 3 compounds, in a buffered medium withmagnesium, support normal germination (9). Pre-incubated spores were then washed and tested in theWarburg apparatus for fermentative capacity withglucose as substrate. Even in complete medium thereis no visible evidence of germination after 3 hours,but it is clear from table VI that fermentative ca-

pacity has been acquired. Preincubation with glu-cose and either ammonium ion or ethanol results ina significant increase in ability of the treated spores

to ferment; incubIation in glucose alone or in saltsolution alone does not cause a significant and re-

peatable increase in fermentative capacity. Controlexperiments showed that neither ethanol nor am-

monium ion added directly to a Warburg flask (withglucose) has any influence on fermentation.

As shown indirectly in figure 3 and 4, ammoniumion accelerates glucose metabolism and in its presence

the oxidation of glucose is more nearly complete thanin its absence. Ethanol has a similar effect on

aerobic glucose metabolism (unpublished data).Since the preincubation (table VI) was aerobic, itappears that acquisition of fermentative capacity fol-lows upon a stimulated aerobic respiration.

Discussion

The contribution of particular carbon atoms ofglucose or other substrates to CO, has been fre-quently used as an index of the balance of competingrespiratory pathways. Cheldelin (8) summarizessome of the most careful and extensive of these de-terminations. In view of our lack of knowledge ofpathways in Fuisariwniii solani f. phaseoli, and in viewof the serious difficulties of interpretation of databased almost solely on CO., labeling (25, 40), we donot propose any conclusion as to the numerical ratioof possible respiratory pathways. Qualitatively, thecurves of C140_ yield from labeled glucose most re-semble published data on Bacillus suibtilis (8), Esch-ericihia coli (37), Claviceps pirpiurea (27), and my-celium of Tilletia caries (29). They differ from anumber of similar measurements made on fungusmycelium or spores in which the output of labelfrom carbon-1 of glucose equals or exceeds that fromcarbons 3 and 4 (6, 18, 30, 37). Surprisingly, ourdata are not at all like those of Heath et al. (19),who found that resting cells of F. lini (F. oxysporumf. lini) convert carbon-1 of glucose to CO, morerapidly than carbons 3 and 4 are so converted.

The interest of the isotope distribution data lies

Table VIThe Development of Aniaerobic Respiratory Capacity

in Ungerminiated SporesUngerminated spores (fresh wt 300 mg) were pre-

incubated 3 hr in a complete medium containing pota s-sium phosphate buffer (pH 6.5) 300 /Amoles, glucose 150,umoles, (NH4) SO4 30 ,umoles, ethanol 75 ,umoles, andMgCl2 10 ,umoles in a total volume of 150 ml, or in defi-cient media as indicated.

The preincubated spores, and untreated controls, werewashed twice by cenitrifugation, suspended in water,filtered, and resuspended for use in the Warburg ap-paratus.

Each Warburg flask contained spores (dry wt 5.3 to8.4 mg, determined individually), potassium phosphatebuffer (pH 6.5) 20 /Amoles, MgCl, 10 ,umoles, 0.2 ml 2 NH2SO4 (in sidearm, tipped at end of experiment), glu-cose 25 ,umoles, and water to 3.2 ml. Flasks were gassedwith prepurified nitrogen and incubated 6 hr; endogenousvalues were corrected for dissolved CO2 and for the(slight) endogenous CO., formation.

Preincubation Q Nc Al/hr/mg

Complete medium 5.7Minus ammonium 2.7Minus ethanol 2.1Minus ethanol and ammonium 0.8Minus ethanol, ammonium, and glucose 0.9None 0.5

539

PLANT PHYSIOLOGY

in the possibility that they may giv\e sonme clue tothe primary respiratory changes associated withgermination and growth. In this respect, it shouldbe noted first that we find that gernmination is markedby a higher proportional conversion of carbon-6 ofglucose, relative to that of carbon-1. to CO., Asmentioned earlier, this is not the case in comparablestudies on bacteria or on grow-ing cells of Clavicepspurptrea (27).

Within the limits of the mlethlo(ds. it is clear thatthe major effects of germination on glucose metabo-lism are essentially duplicated by the provision ofammonium nitrogen to gernminatiing spores. Theeffect is apparent in a short enough tinle so that we

may exclude long-term changes consequent upon

germination, and consider instea(l possible explana-tions based on shifts in equilibria in a miore or lessconstant enzymatic system. Of these, the mlost likelyis that provision of nitrogen accelerates respirationby drawing off intermle(liates for the synthesis ofamino acids; if, for examiiple. glutamiiic aci(d were

formedl from a-ketoglutarate and(l the latter frompyruvate, it is quite reasonable to suppose that an

increase(d formation of glutaniiic aci(d might acceleratethe entire sequence of reactionis lea(ling fronm glucosevia pyruvate to intermediates of the tricarboxylicacid cycle. A second nmechaniismii w-ould also havethe effect of accelerating respirationi: if protein svn-thesis occurs at the expense of ATP. the ADP so

formed may act to accelerate reactions leading fromglucose to pyruvate by acting as a p)hosphate acceptor.The effect of azide on endogenotus respiration ofthese spores (12) suggests that encdogenous respira-tion at least is governed in its rate by the supply ofsuch acceptors.

In terms of known respiratory systems, thechanges in CO. output reporte(d lhere on the addi-tion of amnmonium nitrogen (anld uponI gerimlination)would be explained if amino aci(l synthesis acceleratesthe aerobic oxidation of pyruvate through the tri-carboxylic acid cycle and if the increased rate ofutilization of pyruvate and of ATP in turn acceleratesreactions of the Embden-M\Ieverhof seqluence. Theeffect would be more conv-ersion of carbons 3 and4 of glucose to CO., and a shift of the CJ C,, ratiotoward unity; both are obserxed. The mleclhanismsuggested here has in fact been delmonstrated in yeast

(23), with ethanol formatioin as the indlex ot theactivity of the Embden-MAeyerhof system.

It is unfortunate for logical anld experimentalclarity that the reaction just cited. formi<ation of glu-tamate from a-ketoglutarate. can haive the oppositeeffect on the balance of pathwk-ays (22). in that itprovides TPN, often the limlitinlg factor in operationof the hexosemonophosphate patlhway (7, 21). Forour organism, there is no way of knowing whetheror not TPN limits glucose oxidlation by this pathway;in F. oxysporumn f. lycopersici there are 2 glutamic(lehydrogenases, one using DPN and the other TPN(32); such a system could obviously interconlvert

TPN and DPN so as to maintain the concentrationof oxidized TPN above the critical level.

Respiratory sy-stems or particular componentsthereof often change during spore germination orfrom the spore to the mycelial state (3, 14, 20, 24, 41 ).Sussman, Distler, and Krakow (35) suggest that inascospores of Neuirospora tetrasperina, both the gly-colytic and the Krebs cycle respiratory systeimis (le-velop during the period from activatioln to germlina-tion.

The apparent change in aerobic respiration ofnmacroconidia of F. solani f. phaseoli towa-ar(l one inwhich carbons 1 an(d 6 contribute more nearly equallyto CO., is paralleled by the (levelopmileint of a weakfermentative capacity during the first 3 hours of de-velopment. Since fermlentation is largely base(d onthe Embden-AMeverhof svstem in a relate(d species(10. 19), it is possible to suggest that one chaingeat the cellular level is responsible for both phenonmena.However, it is not yet known wlhether in this speciesanaerobic respiration (loes in fact procee(d hb theEmbden-Mleyerhof reaction sequence.

SummarySugars which support germiinationl are oxi(lize(l

mlore rapidly and wvith less oxidative assinilationthan those hiclh dlo not; germination is accoiml-panied by an increase in the rate of oxidation o0only those sugars whiclh can be utilize(d for germiina-tion. The rate of glucose oxidationi is inverselyproportional to spore (lensity, but the rate of endog-enous respiration andI the extent of utilizatioin ofglucose are invariant with (lensitvy the latter obser-v-ation is interp)retedl as evidence thlat glucose (loesnot suppress or accelerate en(logenouis respiration.Oxygen availability is not the factor responsil)le forthe (lensitv effect. Assimilated glucose is iincor-porate(l primarily inlto a fractioin extractible with80 </,c ethanol, andl the miiajor identifiable comp)oundfornmed is mlannitol. Oxidativelv assimilated glucoseis not appreciably incorlorate(I into cell lipids. Boththe provision of amnmionium nitrogen and germinationresult in characteristic chaniges in the conversion ofspecific carbon atomls of glucose to carbon ldioxidle;it is suggested that these clhanges reflect the accelera-tion of reactions which leadl by wvay of pyrtivate toamino acid synthesis. Ungerminate(d sp)ores )rodluceneither aci(d nor carbon (lioxidle froml glucose aniae-robically; a weak capacity to fermlient gluicose is in-duce(I by germination or b) incubation for a shortperiod in contact with compounds required forgermination.

Literature Cited1. ALLEN, S. H. G., JR. AND D. PowELCSON. 1958a.

Pathways of glucose oxidationi in dividing andnondividing cells of Escherichi(a coli. T. Bacteriol.75: 184-89.

2. ALLEN, S. H. G., JR. AND D. PO\\ELSON. 1958b.Effects of chloramphenicol on glucose oxidationin Escherichia coli. Science 127: 1341-42.

540

COCHRANE ET AL.-METABOLISM IN FUSARIUM

3. BHATNAGAR, G. M. AND P. S. KRISHNAN. 1960.Enzymatic studies on the spores of Aspergillusniger. Part III. Enzymes of Embden-Meyerhof-Parnas pathway in germinating spores of Asper-gillus niger. Arch. Mikrobiol. 37: 211-14.

4. BIDWELL, R. G. S. AND N. R. GHOSH. 1962. Pho-tosynthesis and metabolism in marine algae. IV.The fate of mannitol-C14 in Fuicus vesiculosus.Can. J. Botany 40: 803-07.

5. BONNICHSEN, R. K. AND H. THEORELL. 1952. Anenzymatic method for the micro-determination ofethanol. Scand. J. Clin. Lab. Invest. 3: 58-62.

6. BRANDT, W. H. AND C. H. WANG. 1960. Catabo-lism of C 4-labelled glucose, gluconate, and acetatein Verticilliuiii albo-atrut m. Am. J. Botany 47:50-3.

7. BUTT, V. S. AND H. BEEVERS. 1961. The regula-tion of pathways of glucose catabolism in maizeroots. Biochem. J. 80: 21-7.

8. CHELDELIN, V. H. 1961. Metabolic pathways inmicroorganisms. John Wiley and Sons, Inc., NewYork. 91 pp.

9. COCHRANE, J. C., V. W. COCHRANE, F. G. SIMON,AND J. SPAETH. 1963. Spore germination andcarbon metabolism in Fusaricni solani. 1. Require-ments for spore germination. Phytopathology (inpress).

10. COCHRANE, V. W. 1956. The anaerobic dissimila-tion of glucose by Fusariuml lini. Mycologia 48:48: 1-12.

11. COCHRANE, V. W. 1957. Comparative respiratorymetabolism of spores and mycelium. Phytopathol-ogy 47: 6.

12. COCHRANE, V. W., J. C. COCHRANE, C. B. COLLINS,AND F. G. SERAFIN. 1963. Spore germinationand carbon metabolism in Futsariurni solani. II.Endogenous respiration in relation to germination.Am. J. Botany, (in press).

13. DAWES, E. A. AND W. H. HOLMS. 1958. Metabo-lism of Sarcina lutea. III. Endogenous metabolism.Biochim. Biophys. Acta 30: 278-93.

14. FARKAS, G. L. AND G. A. LEDINGHAM. 1959. Therelation of self-inhibition of germination to theoxidative metabolism of stem rust uredospores.Can. J. Microbiol. 5: 141-51.

15. DE FIEBRE, C. W. AND S. G. KNIGHT. 1953. Theoxidation of glucose by Penticillijin chrysogenumii.J. Bacteriol. 66: 170-72.

16. GIBBS, M., V. W. COCHRANE, L. M. PAEGE, AND

H. WOLIN. 1954. Fermentation of D-xylose-1-C14 by Fusarium lini Bolley. Arch. Biochem.Biophys. 50: 237-42.

17. GOULD, B. S. AND A. A. TYTELL. 1941. Studies inthe physiology of Fusaria. The respiratory andfermentative mechanisms. J. Gen. Physiol. 24:655-61.

18. HEATH, E. C. AND H. KOFFLER. 1956. Biochemis-try of filamentous fungi. II. The quantitative sig-nificance of an "oxidative pathway" during thegrowth of Penzicillium1 chrysogeniuini. J. Bacteriol.71: 174-81.

19. HEATH, E. C., 0. NASSER, AND H. KOFFLER. 1956.Biochemistry of filamentous fungi. III. Alter-native routes for the breakdown of glucose byFusariulm lini. Arch. Biochem. Biophys. 64: 80-7.

20. HOLTON, R. W. 1960. Studies on pyruvate metabo-lism and cytochrome system in Neurospora tetra-sperma. Plant Physiol. 35: 757-66.

21. HOLZER, H. 1959. Carbohydrate metabolism.Ann. Rev. Biochem. 28: 171-222.

22. HOLZER, H. 1961. Regulation of carbohydratemetabolism by enzyme competition. Cold SpringHarbor Symp. Quant. Biol. 26: 277-88.

23. HOLZER, H. AND I. WiTT. Die ersten Stoffwech-selanderung nach Zusatz von NH4+-Ionen zu Glu-cose oxydierenden Hefezellen. Biochem. Z. 330:545-54.

24. IMAMOTO, F., K. IWASA, AND K. OKUNUKI. 1959.The oxidation systems of mitochondrial particlesfrom Aspergillus oryzae. J. Biochem. (Japan) 46:141-50.

25. KATZ, J. AND H. G. WOOD. 1960. The use of glu-cose-C14 for the evaluation of the pathways ofglucose metabolism. J. Biol. Chem. 235: 2165-77.

26. LINDENMAYER, A. 1959. Oxidative metabolism andabsorption spectra of anaerobically grown yeast.Manometric data and absolute absorption spectra.J. Cellular Comp. Physiol. 53: 93-117.

27. McDONALD, J. K., V. H. CHELDELIN, AND T. E.KING. 1960. Glucose catabolism in the ergotfungus Claviceps ptirpurea. J. Bacteriol 80: 61-71.

28. MORRIS, D. L. 1948. Quantitative determinationof carbohydrates with Dreywood's anthrone re-

agent. Science 107: 254-55.29. NEWBURGH, R. W. AND V. H. CHELDELIN. 1958.

Glucose oxidation in mycelia and spores of thewheat smut fungus, Tilletia caries. J. Bacteriol.76: 308-11.

30. NEWBURGH, R. W. AND V. H. CHELDELIN. 1959.Oxidation of labelled substrates by dwarf bunt,Tilletia contraversa. Can. J. Microbiol. 5: 415-17.

31. SANTER, M. AND S. AJL. 1955. Metabolic reactionsof Pasteurella pestis. III. The hexose monophos-phate shunt in the growth of Pasteurella pestis.J. Bacteriol. 69: 713-18.

32. SANWAL, B. D. 1961. Diphosphopyridine nucleo-tide and triphosphopyridine nucleotide linked glu-tamic dehydrogenases of Fusarium. Arch. Bio-chem. Biophys. 93: 377-86.

33. SISLER, H. D. AND COX, C. E. 1954. Effects oftetramethylthiuram disulfide on metabolism ofFusariuml roseum. Am. J. Botany 41: 338-45.

34. SMITH, I. 1960. Chromatographic and Electro-phoretic Technique. Vol I. Chromatography. In-terscience Publishers, Inc., New York. 617 pp.

35. SU'SSMAN, A. S., J. R. DISTLER, AND J. KRAKOW.1956. Metabolic aspects of Neurospora activationand germination. Plant Physiol. 31: 126-35.

36. UMBREIT, W. W., R. H. BURRIS, AND J. F.STAUFFER. 1957. Manometric Techniques. 3d Ed.Burgess Publ. Co., Minneapolis. 338 pp.

37. WANG, C. H., I. STERN, C. M. GILMOUR, S. KLUNG-SOYR, D. J. REED, J. J. BIALY, B. F. CHRISTENSE-N,AND V. H. CHELDELIN. 1958. Comparative studyof glucose catabolism by the radiorespirometricmethod. J. Bacteriol. 76: 207-16.

38. WEST, C. D. AND S. RAPOPORT. 1949. Determina-tion of mannitol and sorbitol in plasma and urine.Proc. Soc. Exptl. Biol. Med. 70: 141-42.

39. WHISTLER, R. AND D. F. DURSO. 1950. Chroma-tographic separation of sugars on charcoal. J.Am. Chem. Soc. 72: 677-79.

40. WOOD, H. G. AND J. KATZ. 1958. The distributionof C14 in the hexose phosphates and the effect ofrecycling in the pentose cycle. J. Biol. Chem. 233:1279-82.

41. ZALOKAR, M. 1959. Enzyme activity and cell dif-ferentiation in Neurospora. Am. J. Botany 46:555-59.

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