Trans. Br. mycol. Soc. 66 (1) 35-43 (1976)

[ 35 ]

Printed in Great Britain

LIPIDS FROM CONIDIA OF ERYSIPHE GRAMINIS TRITICI

(POWDERY MILDEW)

By DENNIS JOHNSON, D. J. WEBER AND W. M. HESS

Department of Botany, Brigham Young University, Provo, Utah 84602

Lipids of conidia of Erysiphe graminis tritici were analysed by thin-layer chromato­graphy, gas chromatography and mass spectrometry. Saturated and unsaturated hydro­carbons (such as Cm Cm C2l: 1> and C25 : 1) and naturally occurring methyl esters of long­chain fatty acids (C17 to C2. ) were detected. The free fatty acids ranged from Cn to C24

and unsaturated fatty acids such as Cn: 1 and C24 : 1 were present. The conidial walls haddifferent appearances when processed with scanning electron microscopy, freeze-etchingand thin-sectioning procedures. Lipid bodies were not present in lipid extracted conidiaand spore walls of lipid extracted conidia characteristically had electron dense tripartitelayers and clumped together.

Powdery mildew fungi infect a large number ofspecies of grains and grasses throughout the world.The conidia of powdery mildew species are uniqueamong the fungi because they germinate at rela­tively low humidity. This germination pheno­menon may be due to the high water content of thespores. For example, the conidia of Erysiphepolygoni contain approximately 70 % water (Yar­wood, 1936,1957)whereas spores from other groupsof fungi contain much less water (Cochrane, 1956).Since powdery mildew conidia germinate in rela­tively dry conditions, fungal lipids may be a factorin preventing desiccation of the spores. Fisher,Holloway & Richmond (1972) investigated fattyacids and hydrocarbon constituents of the surfaceand wall lipids of several fungal spores. They re­ported that the fatty acids from Rhizopus stolonifer,Alternaria tenuis, Botrytis fabae, Neurospora crassaand Penicillium expansum spores were primarilystraight-chained with even numbers of carbonatoms and that the predominant fatty acids werepalmitic and stearic. Polyunsaturated acids werenot detected. The surface hydrocarbons of thespores consisted almost entirely of normal alkanes,but the wall fractions contained complex hydro­carbons. There was an equal distribution of oddand even-numbered carbon alkanes in lipid frac­tions of surface and wall layers. However, Fisheret al. (1972) failed to detect surface lipids from theconidia of E. cichoracearum and E. graminis. Theysuggested that the conidia of Erysiphe were hydro­phobic because of the physical conformation of thesurface structures. Tulloch & Ledingham (1960)analysed the fatty acids of E. graminis by gas chro­matography and found a normal distribution offatty acids. The predominant fatty acids were C16>C18: l' C22: 1> C22, and C24: i- McKeen (1970a, b)

used lipid stains with the light microscope andosmium tetroxide with the electron microscope toinvestigate the lipids in E. graminis hordei. By useof the electron microscope, he detected osmophilicbodies in the conidia and concluded that they werelipids because the stained bodies could be removedwith acetone or alcohol washes. He suggested thatthe water needs for germinating conidia could befulfilled, in part, by the oxidation of fats, sincepowdery mildew spores may germinate in air atlow relative humidity. However, there isno evidencethat such a process occurs. He also suggested thatlipids play an important role in the germinationprocess of conidia of E. graminis hordei. The pur­pose of this study was to investigate the lipid com­ponents that are present in conidia of E. graministritici DC and to observe the effects of lipid solventsupon ultrastructure of conidia.

METHODS AND MATERIALS

Conidia of Erysiphe graminis tritici DC were cul­tured on 'Red Bobs' wheat plants. Conidia werecollected by shaking infected leaves over metal foil.Lipid analyses were performed with two sporesamples which weighted 9'24 g and 11'67 g (freshweight).

The lipids were extracted by adding 50 ml ofspectro-grade n-heptane to the spores for 1h. Next,50 ml of spectro-grade benzene was added to thespore residue for 1 h. Finally, the spores wereextracted by a solution of spectro-grade chloro­form-methanol (2: 1, v/v), and ground in a Sorvallomni-mixer with acid-washed superbrite glassbeads (Minnesota Mining Co.). The ratio of sporesto solution was 3 g/25 ml. The ratio of spores toglass beads was 1 g/1' 5 g. The homogenate was

2-2

Lipids in Erysiphe conidia

heated for 10 min at 45°, then centrifuged at5000 g for 5 min to separate the extract from thespore fragments and beads.

The extracts of all solvents were combined andevaporated to dryness in a nitrogen atmosphere at40°. The total lipid content was determined byweighing the dried residue.

The total lipid residue was separated into in­dividual lipid classes on silica gel G thin-layerplates. The developing solvent system was ben­zene-chloroform (9: 1, v/v). Iodine vapours wereused to visualize the lipid bands. The iodine wasevaporated from the silica gel plates in a hood.Individual lipid fractions (bands) detected by iodinevapours, were scraped from several plates intoseparate beakers. The fatty acid bands were ex­tracted with chloroform and water-saturated diethylether. The hydrocarbon and natural methyl esterfractions of the plates were extracted with benzeneor heptane and water-saturated diethyl ether. Allfractions were suction filtered through fritted glassfilters and reduced to dryness in a nitrogen atmo­sphere at 40°. Esters of the free fatty acids wereprepared by methylating the fractions with BF3

in methanol according to the procedures ofMorrison & Smith (1964). The methyl esters ofthe fatty acids were dissolved in chloroform. Thehydrocarbons were dissolved in n-heptane and thenatural methyl ester fractions were dissolved inbenzene. All lipid fractions were analysed byPackard gas chromatographs (Model 2400) by useof stainless steel or glass columns (3 m x 2 mm ID)packed with 3 % SE 30 on chromosorb Q (1001120 mesh). The temperature programme was50-300° at 2° or 4° per min. The nitrogen flow ratewas 35 ml/min.

Hydrocarbons, fatty acids, and naturally occur­ring esters of fatty acids were identified by com­paring retention time with known standards. Pre­liminary identification based on GC retention timewas verified by use of the gas chromatograph-massspectrometer (Varian MAT 111) by use of a glasscolumn packed with 1 % SE 30 on chromosorb Q(100/120 mesh). The temperature programme was50-300° at 2° or 4° per min. The helium flow ratewas 15 ml/min. The scan conditions were 100 tu]sec; the ion source temperature was 200 0 and theionization energy was 80 eV. The individual spectrawere identified by interpretation of the mass frag­ments and by submission of the mass fragmentsand intensity values to a computer library inBethesda, Maryland (NIH mass spectral system),by means of a teletype connected to a telephone.

Lipids were extracted from conidia for electronmicroscopy investigations by immersion in heptanefor 1 hat 500, then benzene-chloroform, 3: 1 for 1 hat 50°, For transmission electron microscopy of

thin sections, conidia were processed according tothe procedures described by Hess (1966) exceptthat dehydrated tissues were embedded in Spurr(1969) resin. Freeze-etch replicas were preparedaccording to the methods described by Sassen,Remsen & Hess (1967) except that replicas werecleaned in sulphuric acid-dichromate cleaningsolution for 6-12 h (Hess, Bair & Neushul, 1972).For scanning electron microscopy investigationsconidia were critical point dried to prevent con­formational changes then coated with evaporatedgold while they were rotated through 360°.

RESULTS AND DISCUSSION

Based upon spore wet weight, the spores ofpowdery mildew contained 5'4 % lipid. The lipidextract was separated into seven bands by use ofthin-layer chromatography. Individual bands froma large number of thin-layer plates were combinedand the lipid was eluted from the silica gel G withspectrograde solvents, after which the individualfractions were analysed by gas chromatography.

The major mass fragment values are listed intable form along with the names of the compoundsidentified (Tables 1-3). The Tables also containthe relative percentage composition based on theareas of the individual peaks in the respective lipidgroups.

The hydrocarbons detected are listed in Table 1.

The hydrocarbon chain length ranged from C16 toC25 and the odd chain length was predominant.The hydrocarbons were mainly unsaturated.

The naturally occurring methyl esters detectedare listed in Table 2. The mass fragments andintensities were characteristic of methyl esters offatty acids. The chain length ranged from C16 toC25 •

The fatty acids are listed in Table 3. Althoughthey were methylated in order to analyse them byGC, they are listed as the acid form. The range wasfrom Cn to C23• A rather wide distribution of evenand odd chain fatty acids was present.

Possible sources of the hydrocarbons are fromthe waxy surface coating or from within the cellwalls of the conidia. Fisher et al. (1972) prepareda cell wall fraction and failed to detect lipids in thecell wall fraction. This suggests that the hydro­carbons in our analyses came from inside the cell,although Fisher's sample size was approximatelyone-fifth our sample size and may have beenbelow the hydrocarbon detection level.

The alkenes were predominantly of odd chainlength, which is consistent with previous reportsof spore hydrocarbon analyses (Weete et al, 1969).The saturated hydrocarbons were also predomi­nently odd chained compounds.

D. Johnson, D. J. Weber and W . M. Hess 37

Table 1. Hydrocarbons detected in lipid extracts of conidia of Erysiphe graminis tritici

(Only the major ion fragment s are listed. The relative composition is based on the area of the gas chromatographicpeaks of the hydro carbons.)

Relative %M/E and intensity of ion fragments Compounds Formula composition

55 59 83 97 224* Hexadecene C16 : 1Ha2 2'755 69 83 97 238* Heptadecene C17 : 1Ha• 21 '2

GC retentive time identification Octadecene C18:1Ha6 6'557 71 85 97 113 268* Nonadecane C19H.o 2'9

GC retent ive time ident ification Nonadecene C19 : tHa8 21 '055 67 81 95 294* possible diene C21:2H.o 15'5

100% 100% 82% 64% 7 %41 55 69 83 97 Cycloalkene C21 : tH.. 5'0

100% 87% 58% 33% 28%GC retentive time identification Heneicosene C21:1H.2 0'7GC retentive time identification Docosene C2~:tHH 0'2

55 69 83 97 111 125 322* Tricosene C2a:tH.6 11'995% 44% 3% 29% 16% 21% 1%41 55 69 81 95 109 320* 3,12-Tricosadiene C2a:2H.. 2,891% 100% 55% 33% 24 % 17% 2%

GC retentive time identification Tetracosene C24: tH.8 0'7GC retentive time identification Pentacosen e C2S:tHSO 8,8

* Parent ion.

Table 2 . N aturally occurring methyl esters of fatty acids detected in lipid extracts of conidia of Erysiphegraminis tritici

(Only the major ion fragments are listed, The relative percentage composition is based on the area of the gaschromatographic peaks of the natural methyl esters of the fatty acids.)

Relative %M/E and intensity of ion fragments Compounds Formula composition

74 87 149 180 222 254* Methyl pentadecenoate] Ct6:tHa202 1'175% 31% 100% 6 % 6 % 5 %74 87 227 239 270* Methyl hexadecanoatej Ct,H a.0 2 0'367% 17% 8% 6 % 6 %74 87 143 199 227 239 270* M ethyl hexadecanoatej C1,H8602 23'3

100% 88% 13% 10% 10% 11 % 9 %74 87 143 199 311 354* Methyl docosanoate] C23H.602 22'388% 56 % 14% 4 % 4 % 7 0 1

/ 0

71 74 87 278 320 352* Methyl docosenoatef C2a:tH'602 10'260% 40 % 34% 4 % 4 % 4 %74 87 143 339 382* Methyl tetracosanoatet C2sHs202 10'780% 77% 20% 3% 5 %69 74 87 147 348 380* Methyl tetracosenoate C2s :tHso02 32'142 % 18% 42 % 10% 4% 3 %

* Parent ion,t Natural methyl ester of fatty acid.

Naturally occurring methyl or ethyl esters offatty acids have been reported for Rhizopus arrhizus(Weete et al. 1970), Ustilago maydis (Laseter, Weete& Weber, 1968), and Tilletia species (Laseter,Hess, Weete, Stocks & Weber, 1968). The naturalesters of fatty acids may be parts of membrane com-

plexes or they could be reserve energy sources inequilibrium with free fatty acids,

The longer chain lengths of the fatty acids are ofparticular interest as most fatty acids in fungalspores have CIS or CIS chain lengths, whereas inconidia of powdery mildew, fatty acids with longer

38 Lipids in Erysiphe conidia

Table 3. Fatty acids present in lipid extracts of conidia of Erysiphe graminis tritici

(The extract was methylated before gas chromatograph-mass spectrometer analysis. The relative percentagecomposition is based on the area of the gas chromatographic peaks of the fatty acids.)

Relative% com-

M/E and intensity of ion fragments Compounds Formula position

Retention time Dodecanoic acidj CllHsaCOOH Trace(Lauric acid)

74 87 143 185 197 228* Tridecanoic acidj C13HssCOOH 0'446% 18% 15% 17% 4% 5%74 87 143 199 211 242* Tetradecanoic acid] C13HS7COOH 4'0

100% 64% 14% 9% 5% 3% (myristic acid)

74 87 143 213 225 256* Pentadecanoic acid] C14Hs9COOH 0'4100% 69% 11% 5% 4% 6%

74 87 105 180 222 254 Pentadecenoic acidj CUHS7COOH Trace52% 38% 68% 9% 5% 6%

74 87 143 227 239 270* Hexadecanoic acidj C1sH31COOH 6·8100% 71 % 14% 8% 4% 6% (Palmitic acid)

55 74 87 194 236 268* cis-c-Hexadecenoic acidf Cls:lHa9COOH 0'1100% 24% 26% 15% 12% 9%74 87 143 241 255 284* Heptadecanoic acidj C16HaaCOOH Trace

100% 71 % 13% 10% 67% 8%

74 87 208 250 282* Heptadecenoic acidj C16:1H31COOH 1'732% 20% 4% 4% 3%74 87 143 199 225 267 298* Octadecanoic acidj C17HasCOOH 2'7

100% 71 % 10% 10% 6% 3% 8% (Stearic acid)

67 74 81 87 220 262 294* cis-q-cis-tz-Octadecadienoic c., :sHalCOOH 1·8100% 20% 63% 20% 3% 3% 7% acidj (Linoleic acid)

74 87 143 199 269 281 321* Nonadecanoic acidj C16Ha7COOH 6'0100% 91 % 18% 4% 6% 3% 8%

71 74 87 99 236 278 310* Nonadecenoic acid'] C16:1H.sCOOH 5'667% 17% 20% 78% 6% 6% 3%55 74 87 283 326* Eicosanoic acidf C1.Ha9COOH 10'789% 28% 40% 3% 3% (arachidic acid)

43 74 87 250 292 324* cis-c-Eicosenoic acidt C19 :1Ha7COOH 0'5100% 21% 42% 3% 3% 2% (gadoleic acid)

55 74 87 297 209 340* Heneicosanoic acid'] CaoH41COOH 4'574 87 143 199 311 323 354* Docosanoic acid'] C21H4.COOH 15'3

100% 61 % 20% 5% 5% 7% 6% (behenic acid)

55 74 87 278 320 352* cis-rg-Docosenoic acidj Cal:1H41COOH 12'198% 41% 54% 5% 5% 5% (erucis acid)

74 87 143 199 339 351 382* Tetracosanoic acid] CaaH47COOH 15"3100% 69% 19% 5% 5% 3% 5% (lignoceric acid)

55 74 87 113 306 348 380* cis-r 5-Tetracosenoic acidj Ca.: IH4SCOOH 11'5100% 49% 77% 5% 3% 9% 5% (nervonic acid)

* Parent ion. t Free fatty acid.

chain length, up to Ca3 are present. Long-chain the long-chain fatty acids may be implicated asfatty acids have also been reported for yeasts by hormonal or regulatory agents.Welch & Burlingame (1973), who suggested that The longer fatty-acid chain lengths of powderythe fatty acids with chain lengths longer than mildew spores may also be related to the ability18 carbons had important metabolic roles such as of these spores to retain relatively high amounts oftransport in membranes or involvement in biologi- water in dry environments.cal control mechanisms. They also suggested that The most likely function of the hydrocarbon

D. Johnson, D. J. Weber and W. M. Hess

Figs. 1-7 are of Erysiphe graminis tritici conidia. Figs. 1-2 are scanning electron micrographs of conidiacoated with gold, fig. 3 is a freeze-etch replica of a conidial surface, and figs. 4-7 are thin sections ofportions of conidia.

Fig. 1. Developing conidia. x 1350.

Fig. 2 . Conidial surface. x 12600.

39

4° Lipids in Erysiphe conidia

w

Fig. 3. Conidial wall. x 9500.

Fig. 4. A portion of a conidium showing the spore wall (CW) and lipids (L). x 34000.

D. Johnson, D.J. Weber and W. M . Hess

Fig. 5. Portions of conidia which were treated with lipid solvents. x 13000. Note that lipids are notevident inside conidia and conidia stick together (see arrows).

41

Lipids in Erysiphe conidia

Fig . 6. A portion of a conidium which was not treated with lipid solvents showing the uniform granularityof the cytoplasm, a characteristic lipid body (L) and the spore wall (CW) with the characteristic electron­dense outer layer. x 24000.

Fig. 7. A portion of a conidium which was treated with lipid solvents showing the disrupted cytoplasm,absence of lipid bodies and the spore wall (CW) with a tripartite electron dense layer on the surface.x 24000.

compounds appears to be as a water barrier withinthe wall or as a component of spore wall surfaceswhich reduces water loss. Erysiphe graminis triticiconidial surfaces have irregular ridges (Figs. 1, 2)when examined with scanning electron microscopy,however, freeze-etch replicas of conidial wallsreveal a blocky structure (Fig. 3), which indicatesthat powdery mildew conidial walls are not con­structed like other fungal spore walls examined todate (Hess, 1973). It is possible that part of thewall is fractured away during the freeze-etchingprocess. Also, few lipid bodies are observed inthin sections of dormant conidia (Figs. 4, 6), whichsuggests that the lipoidal components discussedabove are not entirely from the cytoplasm, althoughlipid bodies are not present in conidia from whichthe lipids have been extracted (Figs. 5, 7). Also,the surfaces of conidia are significantly changedby lipid extraction procedures since lipid extractedconidia clump together and appear to be attachedby fibrous strands (Fig. 5). Surfaces of lipid

extracted conidial walls also characteristically havean electron-dense tripartite layer (Fig. 7) whichis not present on non-extracted conidia (Fig. 6).Ultrastructural investigations therefore indicatethat lipids are extracted from the cytoplasm andthat the spore walls are significantly altered by thelipid extraction procedures.

Further investigations are necessary to deter­mine whether the longer chain length fatty acidsare a factor in survival under dry conditions.Analyses of conidia of other powdery mildew fungishould be conducted to see whether longer chainlength fatty acids are present.

This research was supported in part by NIHResearch Support Grant, administered by theResearch Division of the Brigham Young Univer­sity, by NSF Grant GC29503, and by NationalInstitutes of Health Career Development AwardNo. 1-K4-GM, 976 to W. M. Hess.

D. Johnson, D. J. Weber and W. M . Hess 43

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HESS, W. M. (1966). Fixation and staining of fungushyphae and host plant root tissue for electronmicroscopy. Stain Technology 41, 27-35.

HESS, W. M. (1973). Ultrastructure of fungal sporegermination. Shokubutsu Byogai Kenkyu (Forsch.Gebiet P/lanzenkrankh.) Kyoto 8, 71-84.

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WEETE, J. D., LASETER, J. L ., WEBER, D. J., HESS,W. M . & STOCKS, D. L. (1969). Hydrocarbons, fattyacids, and ultrastructure of smut spores. Phyto­pathology 59, 545-548.

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(Accepted for publication 19 May 1975)