Sterolsland the Production of Oospores by Phytophthova cactovum

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1 Journl of Generl Microiology (I972), 72, Printed in Gret Britin 321 Sterolslnd the Production of Oospores y Phytophthov cctovum By C. G. ELLIOTT Deprtment of Botny, The University of Glsgow, Glsgow, G12 8QQ (Accepted for puliction 26 April 1972) SUMMARY The effect of eight sterols on oospore production y Phytophthor cctorum hs een studied quntittively. Their ctivity ws found to e in the following order:,hitosterol= A5-vensterol = fucosterol = stigmsterol > cholesterol = 7-dehydrocholesterol > ergosterol > cholestnol. INTRODUCTION The oomycete fungus, Phytophthor cctorum (Le. & Cohn.) Schroet., together with other species of Phytophthor nd Pythium, is unle to synthesize sterols. It cn grow vegettively in the sence of sterol, ut for the development of sexul reproductive orgns (oogoni nd ntheridi) nd for the development of oospores from the fertilized oogoni, sterols must e present in the medium. Differences etween sterols in ility to promote oospore formtion in P. cctorum were descried y Elliott, Hendrie & Knights (1966). They reported tht P-sitosterol nd A5-vensterol (29-isofucosterol) were highly ctive, cholesterol nd ergosterol less so, while with fucosterol very few oospores were found. This difference etween vensterol nd fucosterol, the two isomers of 24-ethylidenecholesterol, ws remrkle, so more detiled study of the ctivity of these nd other sterols hs een mde. The following hve een studied : cholestnol (g-cholestn-3p-01); cholesterol (cholest-5-en-3/3-01); 7-dehydrocholesterol (cholest-5,7-dien-3~-01) ; ergosterol (24R-methylcholest-5,7,(E)22-trien-3P-o1) ; p-sitosterol (24R-ethylcholest-5-en-3P-01) ; A5-vensterol (2~-ethylcholest-~,(Z)2~(28)-dien-~,8-ol) ; fucosterol (24-ethylcholest-5,(E)24(28)-dien- 3p-01); stigmsterol (24S-ethylcholest-5,(E)22-dien-3/3-01). METHODS Phytophthor cctorum (IMI 21168) ws grown t 24 "C in the drk on th sucrosesprgine medium descried y Elliott et l. (1966) nd Elliott (1968) to which clcium chloride (0.1 gfl) ws dded. The medium ws solidified with I % (wlv) Difco Bcto-gr. The medium ws dispensed in 25 ml lots in IOO ml flsks nd utoclved; the sterol ws then dded to the flsks in ether solution, nd five Petri dishes (5 cm dim.) poured from ech flsk. Ech dish ws inoculted centrlly with disc cut from culture growing on sterol free medium. The ctivity of the sterol ws ssyed y counting oospores in strip trnsect from the edge to the centre of the colonies y viewing single plne of focus through the ottom of the dish (Elliott, 1968). It ws not prcticle to study ll sterols in single experiment; therefore series of experiments ws crried out, in ech of which cholesterol ws included s stndrd. The cholesterol ws commercil smple purified y chromtogrphy on lumin followed y recrystlliztion from methnol. Stigmsterol ws commercil smple recrystllized from methnol. Both compounds were pure on the evidence of gs-liquid 22 MIC 72 Downloded from y

2 322 C. G. ELLIOTT chromtogrphy (g.1.c.). Fucosterol ws extrcted from thlli of Ascophyllum nodosum nd purified y chromtogrphy on lumin nd recrystlliztion from methnol. Its purity ws verified y g.1.c. nd it ws found to hve trce of compound presumed to e clionsterol (24s-ethylcholesterol). p-sitosterol ws extrcted from seed of Aven stiv nd ws pproximtely 85 % pure, the principl contminnts eing cholesterol nd cmpesterol (24-methylcholesterol). Avensterol ws extrcted from seed of Lepidium stivum nd purified y chromtogrphy on lumin nd on silver nitrte-silic gel fter cetyltion, followed y recrystlliztion of the cette nd hydrolysis; it hd only trce impurities. Ergosterol nd 7-dehydrocholesterol were commercil smples (Sigm Chemicl Co.). Both hd stisfctory ultrviolet sorption spectr, nd the 7-dehydrocholesterol ws colourless. Exmintion of comprle smple of ergosterol from the sme source y g.1.c. on OV-17 showed trces of the relted diene sterols nd mrked shoulder to the min pek. When such ergosterol smples were suject to eril oxidtion, the min pek ws lost nd pek remined with the retention time of the shoulder of the originl pek. The cholestnol (from Sigm Chemicl Co.) ws t lest 99% pure (see Elliott, 1968). For nlysis of vrince, the individul counts, x, were trnsformed to J(x + 4). Comprison of the effect of one sustnce in different experiments or of different sustnces in one experiment ws mde y plotting curves relting oospore numer to sterol concentrtion, nd exmining (i) the verge heights of the curves ove the horizontl xis, nd (ii) their similrity in slope nd curvture. Differences in height re shown in the nlysis of vrince y significnt min effects, nd differences in slope or curvture y significnt interctions. RESULTS The counts for cholesterol vried from one experiment to nother (Tle I, Tle ZQ), ut replictions within experiments were not significntly different. The numers of oospores formed declined stedily with time from Expts I (done in Mrch 1970) nd 2 (August 1970)~ through Expt 3 (June 1971) until n experiment done in July 1971 gve such low fertility tht the results hd to e discrded. The fungus ws frequently sucultured during this time on sl medium, new inocul eing tken s rule from the edge of the colony. An old culture ws revived nd used in Expt 4 (August 1971). It proved much more fertile, ut it ws noted tht its hyphl growth rte ws only out hlf tht of the previously used line. The revived line ws used in Expt 5 (Novemer 1971) ; in this experiment fertility ws slightly lower thn in Expt 4. The four CZ9 sterols, p-sitosterol, fucosterol, A5-vensterol nd stigmsterol, hd similr ctivities, s shown y the results for Expts I nd 2, compring,8-sitosterol nd fucosterol, Expt 3, compring stigmsterol nd vensterol, nd Expt 5, compring fucosterol nd stigmsterol (Tle I). The nlysis of vrince (Tle 2) showed no significnt min effects for differences etween sterols, lthough in Expt 5 (Tle 2e) the men squre for the difference etween fucosterol nd stigmsterol ws rther lrge; however it ws not significntly greter thn tht for replictions, or even for tht for replicte Petri dishes lthough the ltter ws itself rther lrge. In Expts I nd 3 the effect of concentrtion ws different for the different sterols, giving significnt Interction I (Tles 2 nd d). In Expt I, the mximum count for p-sitosterol ws t 4 mg/l, for fucosterol t 25 mg/l. In Expt 3, the count for vensterol t ~onig/l ws peculirly low. However, in oth experiments the differences were smll nd in Expts 2 nd 5 Interction I ws not significnt. Vrition etween replicte Petri dishes ws lwys significntly greter thn tht etween individul counts within dishes (Tle 2). Downloded from y

3 Sterols nd spores of Phytophthor Tle I. Men counts of oospores of Phytophthor cctorum produced in medi with vrious sterols Ech viue is the men of 4 counts in ech of 5 Petri dishes, or occsionlly of 5 counts in ech of 4 dishes. nd : replictions mde y supplementing sl medium from two independent seril dilutions of common stock solution of the sterol. Sterol concentrtions (mg/l) Experiment I Sterol Cholesterol 8-Sitosterol Fucosterol 2 Cholesterol P-Si tos terol Fucosterol Ergosterol 3 Cholesterol Stigmsterol A5-Avensterol 4 Cholesterol 7-Dehy drocholesterol 5 Cholesterol Fucosterol Stigms terol Cl n CC C1 n ' '3 I 6.8 I I 38.0 I 8.5 I 2-7 I 6.9 9' ' '0 I I I 69. I I0 25'7 32'5 43' '3 I '9 I 1.4 I '5 42' '8 85'9 75' '4 54' '9 15' ' ' ' '3 9'5 11' '4 67'5 55'3 73'6 44' '2 I 08.6 A '8 37' '4 23' '2 35' '2 7'3 I I '2 I ' '5 5'3 24' ' '3 I '0 2'1 2'9 3'4 3' I ' '3 27' ' \ ' '5 9'2 2' '4 2' ' ' I '4 2' '4 4'1 7.6 The numer of oospores formed with fucosterol ws not ffected y ddition of clcium chloride to the medium (Tle 3). The ctivity of the C29 sterols ws considerly greter thn tht of cholesterol. Their reltive ctivities were exmined y plotting the pooled oospore count for the CZ9 sterols ginst tht for cholesterol for equivlent sterol concentrtions in ech experiment (Fig. I). Expt 2 ws unusul in tht the count for cholesterol declined t the two highest concentrtions, nd these two points were omitted from n nlysis which showed no significnt differences etween experiments in the regression of the C29 sterol count on the cholesterol count, the joint regression coefficient eing I -67. Thus despite differences in fertility etween experiments, the numer of oospores (mesured on the squre-root scle) formed in response to certin quntity of one of the C29 sterols ws 1.67 times the numer formed in response to the sme quntity of cholesterol. The ctivity of ergosterol ws significntly less thn tht of cholesterol t the lower concentrtions (Tle I, Expt 3). Cholesterol nd 7-dehydrocholesterol hd the sme ility to promote oospore formtion in Phytophthor cctorum (Tle I, Expt 4). " Downloded from y

4 Tle 2. Anlysis of vrince of oospore counts Interction I is tht due to differences etween oospore counts t different concentrtions in the vrious experiments, or with the vrious sustnces. Interction 2 is due to differences in the count t different concentrtions etween the replictes nd. Concentrtions Expts or sustnces Interction I Replictions nd Interction 2 Petri dishes Counts within dishes 7 7 C Expts I to 5 Expt I Expt 2 - Cholesterol P-SitosterollFucosterol P-SitosterollFucosterol 7- Degrees of Degrees of Degrees of freedom Men sq. freedom Men sq. freedom Men sq ' I '79" I 13'49 I '34" " NS 2 23'95t NS NS " " " 824 1' I *so d EXPt 3 Stigmsterol/Avensterol Degrees of freedom Men sq I 96 I I -08 * Significnt t I % level; -1 significnt t 5 % level; NS, not significnt. 5 I 2-72" I 0'01 5 9'55 NS " 273 I -65 e EXPt 5 Stigmsterol/Fucosterol h I-- 7 Degrees of freedom Men sq. I NS '10 5 5' NS I0 I '02" '07 Downloded from y

5 Sterols nd spores of Phytophthor P P 325 A I 1 I 1 I Spore count (cholesterol) Fig. I. Reltionship etween oospore counts for equivlent concentrtions1of the CZ9 sterols nd cholesterol..---, Expt I; x-x, Expt 2; A----A, Expt 3; 0--0, Expt 5. Tle 3. Comprison of oospore production y Phytophthor cctorum in medium with fucosterol nd with, nd without, dded clcium chloride (0.1 gll) Ech vlue is men of 20 counts. Fucosterol concentrtion (mg/l) A r I 0.25 With CCI, Without CCI, It ws shown previously (Elliott, 1968) tht the numer of oospores produced with cholestnol is much lower thn with cholesterol. However, considerle numers of oogoni re formed in cultures grown in cholestnol, ut mny fil to develop into oospores (Tle 4). Whether the C29 sterols differ from cholesterol in the numers of oogoni produced or in the numers of oospores resulting from similr numers of oogoni hs not een investigted quntittively, ut from inspection of developing cultures it ppers tht more oogoni re produced with the C29 sterols. Downloded from y

6 326 C. G. ELLIOTT Tle 4. Men counts of oogoni nd oospores produced in medi witji cholesterol or cholestnol The numer of oogoni were scored t 10 dys. The siiie dishes were scored gin t 21 dys for numers of oospores. Sterol concentrtion (mgjl)?--l I 0.25 Oogoni Cholesterol Cholestnol ~ Oospores Cholesterol Cholestnol o DISCUSSION The vrition in numers of oospores in these experiments is of severl kinds. There is considerle vrition in the count from one prt of dish to nother, nd lso significnt differences etween the replicte Petri dishes. Differences etween dishes re ffected prticulrly y vrition in thickness of the medium. With thick lyer of medium there were fewer oospores ginst the ottom of the dish thn with thin lyer. Vrition etween the replicte dilution series (: nd, Tle I) ws generlly not significnt. There were lso differences etween different experiments, presumly the result of genetic vrition in the fungus. Proly s result of the method of suculturing the fungus, selection for incresed growth rte occurred, nd ssocited with this ws decline in fertility. These differences, however, do not oscure the vrition ttriutle to differences in ctivity etween the sterols. The sterols fll into the following order in their ility to promote oospore formtion' in Phytophthor cctorum : p-sitosterol = fucosterol = A5-vensterol = stigmsterol > cholesterol = 7-dehydrocholesterol> ergosterol > cholestnol. There re two importnt differences from our previous results (Elliott et l. 1966). Fucosterol, previously regrded s of low ctivity, hs een found to e highly ctive, with ctivity equl to tht of vensterol, nd 7-dehydrocholestero17 previously reported s inctive, hs een found equl in ctivity to cholesterol. These differences cnnot e explined stisfctorily, ut we hve pid more ttention thn efore to the purity nd correct identity of the sustnces tested nd to the quntifiction of their ctivity. The ddition of clcium chloride to the medium in the present experiments ws not the reson for the different result with fucosterol. The ctivity of the CZ9 sterols compred to the C,, sterols (cholesterol nd 7-dehydrocholesterol) shows the importnce of the two extr cron toms t cron 24 in the side chin, ut the precise configurtion with respect to the doule ond C-24(28) does not pper to e importnt. The reduced ctivity of ergosterol t low concentrtion s compred with cholesterol is proly not due to the second doule ond in the B ring, ecuse 7-dehydrocholesterol hs the sme B ring configurtion s ergosterol nd the sme ctivity s cholesterol. 7-Dehydrocholesterol ws found to hve comprle ctivity to cholesterol in Pythium periplocum nd P. proltum y Dr 6. W. Hendrix (personl communiction, 1971). Ergosterol hs doule ond in the side chin t cron 22, ut so hs the highly ctive stigmsterol. On the other hnd, the methyl group t cron 24 of ergosterol hs the /3-configurtion, while the 24-ethyl group of stigmsterol nd /3-sitosterol hs the opposite -configurtion (Fig. 2). This, however, my not ccount for the difference in ctivity Downloded from y

7 Sterols nd spores of Phytophthor 327 Fig. 2. Structure of stigmsterol, giving the numering of cron toms nd lettering of rings. This shows the -configurtion of the 24-ethyl group common to P-sitosterol nd stigmsterol; ut note tht the 24-methyl group of ergosterol hs the opposite &configurtion. etween these compounds since Hendrix finds tht in Pythium cmpesterol (which hs n z-methyl group t C-24) nd rssicsterol (with P-methyl group t C-24 s well s doule ond t C-22) re oth highly ctive. (The structure of these sterols ws reviewed y God (1967).) The low ctivity of ergosterol my e due to its decomposition to n inctive sustnce efore it cn e tken up y the fungus, or to some effect of the trce comtminnts. I m indeted to Dr B. A. Knights for prepring nd nlysing the sterols. REFERENCES ELLIOTT, C. G. (1968). Competition nd synergism etween cholesterol nd cholestnol in oospore formtion in Phytophthor cctorum. Journl of Generl Microiology 51, I ELLIOTT, C. G., HENDRIE, M. R. & KNIGHTS, B. A. (1966). The sterol requirement of Phytophthor cctorum. Journl of Generl Microiology 42, GOAD, L. J. (1967). Aspects of phytosterol iosynthesis. In Terpenoids in Plnts, pp Edited y J. B. Pridhm. London nd New York: Acdemic Press. Downloded from y