BURROUGHS, R., SEITZ, L. M., SAUER, D. B. & MOHR,H. E. (1976). Effect of substrate on metaboliteproduction by Alternaria alternata. Applied Environ-mental Microbiology 31, 685-690.

GATENBECK, S. & SIERANKIEWICZ, J. (1973). Microbialproduction of tenuazonic acid analogues. Anti-microbialAgents Chemotherapeutics 3, 308-309.

HARVAN, D. J. & PERO, R. W. (1976). The structureand toxicity of the Alternaria metabolites. Advancesin Chemistry Series 149, 344-355.

HARWIG, J., SCOTT, P. M., STOLTZ, D. R. & BLANCH-FIELD, B. J. (1979). Toxins of molds from decayingtomato fruit. Applied Environmental Microbiology38, 267-274.

HULT, K. & GATENBECK, S. (1976). Regulation andmetabolic background of polyketide formation. I.Effects of (- )-hydroxycitrateand metabolicroles ofcitrate and malate in fatty acid and polyketideformations. Acta Chemica Scandinavica B 30, 283-286.

HULT, K. & GATENBECK, S. (1978). Production ofNADPH in the mannitol cycle and its relation topolyketide formation in Alternaria alternata. Euro-pean Journal of Biochemistry 88, 607-612.

HAGGBLOM, P. & UNESTAM, T. (1979). Blue lightinhibits mycotoxin production and increases totallipids and pigmentation in Alternaria alternata,Applied Environmental Microbiology 38, 1074-1077.

Notes and briefarticles

modified CD-medium (1 +1) at no dilution ordiluted 1/100 stimulated toxin production inboth the low-producing and high-producingstrains. Rice extract gave toxin levels in the low-producing strain, after seven days, equivalent tothose of the high-producing but without anychange of morphology (Haggblom, unpubl.),Transfers from the low-producing strain that wascultivated on modified CD-medium: rice extract(1 + 1) to modified CD medium resulted in a returnto low levels of toxin production. A continuoussupply of rice extract is then necessary for a highAOH and AME production in the low-producingstrain.

The results from the rice-extract experimentsshowed that there was no absolute requirement foran AOH- and AME-producing strain to be highlybranched as the extract was able to increase toxinproduction to a 'normal' level without any changein morphology.

In the low-producing strain it is possible thatthe metabolic pathway yielding AOH and AMEis blocked, and compound(s) from the rice will thenincrease toxin production by supplying inter-mediates for the synthesis.

This work was supported by the SwedishCouncil for Forestry and Agricultural Research.

REFERENCES

EFFECT OF LIGHT (ESPECIALLY NEAR-U.V.) ON SPORE GERMINATIONAND ULTRASTRUCTURE OF VERT/CILLIUM AGARICINUM

MOHAMED OSMAN AND L. R. G. VALADON

Department of Botany, Royal Holloway College,University of London, Egham Hill, Egham, Surrey TWzo oEX

Growth, sporulation and germination of spores offungi may be influenced by various light (irradia-tion) conditions. Zadoks & Groenewegen (1967)found that white light (7600 lx) inhibited thegermination of urediniospores of wheat brownrust. Also Harda (1975) showed that light inhibitedconidial germination and germ-tube growth ofMonilinia mali to a certain extent. On the otherhand Berg & Gallegly (1966) reported that oosporesin species of Phytophthora failed to germinate after30 days in darkness but germinated when exposedto fluorescent light for 7 days, blue and far-redirradiations being the most effective in producingthe highest percentage of germination. More

recently Schneider & Murray (1979) have foundthat germination of Puccinia graminis uredinio-spores was induced by 1 min red irradiation at660 om, and this induction could be reversed by1 min far-red irradiation at 730 om. They sug-gested the presence of a phytochrome system inthis fungus. Osman & Valadon (1979) have shownthat the effect of various irradiations on growth andsporulation of V. agaricinum was variable and notlight-dependent. Furthermore, they also showedthat phytochrome may be involved in caroteno-genesis in this fungus (Valadon, Osman & Mum-mery, 1979), while some other photoreceptor(s)in the near-u.v, might be responsible for increased

Trans. Br. mycol. Soc. 77 (1), (1981). Printed in Great Britain

188 Notes and briefarticles

Table 1. Effect of different irradiations on spore germination of V. agaricinumas a function of time

Percentage spore germinationat various times (h)Irradiation and transmittance

(nm) 9 11 13 15 17 22Dark 11'8±3'9 18'7 ±1 '9 36'1±9 '2 47'6±6'6 63'1±2'8 82'2±4'1White (visible spectrum) 4'5 ±2'7 13'2±4'5 33'1± 2'4 39'2±7'0 55'2±5'6 68'6±4'1Black (300-420, peak 355) 3'7± 1"8 10'1 ± 3'0 19'9±2'7 27'5±2'6 6o,6±7'O 72'0±3'lNear-u.v, (320-45°, peak 370) 0'7 ± 1'4 8'2 ±2'9 11'2±3'0 16,8±2'0 20'5± 3'0 28'3 ±3 '7Blue (380-525, peak 473) 10'9±4'6 15'1± 3'6 31'7±4'7 43'9±7'1 54'2±3 '4 72'8 ±3'3Yellow (545-620,peak 520) 11'1±3'9 21'6±2 '4 30'5 ± 4'5 44'3±4'3 57'S±3 '7 69'9±3'9Red (625 and above) 7'4±2'8 21'9 ±2'8 38'7±4'7 49'9±6'9 62'6±3'7 81'2 ±2 '2

carotenoid synthesis. Since near- u.v. had such adramatic effect on the appearance of this fungus(Osman & Valadon, 1979), the effects of near-u.v.and other irradiations (Table 1) on the germinationof spores and hyphal ultrastructure were inves-tigated.

Petri dishes containing 20 ml of 2 % solid maltagar were divided into squares of approximately1 em, one drop (0'05 ml) of a spore suspensionfrom 7-day-old dark-grown V. agaricinum (Lk)Cda (Valadon & Mummery, 1971) cultures wasplaced on each square and the Petri dishes wereincubated at 24-25 °C under different irradiations(Table 1). Ilford filters (662 blue), 626 (yellow)and 608 (red) were used. For black irradiation 2fluorescent tubes (Philips TL 40 W/ 08 RS, F 40BLB) and for near-u.v, 2 fluorescent tubes(Philips 4 FT 40 W/05 ) were used. The fungalcultures were irradiated for various times toreceive a radiance of approximately 1 W m -2. Theresults (Table 1) show that near-u.v. irradiationsignificantly decreased spore germination whichwas only 28'3 %, while the dark-germinatedspores reached a percentage of 82 %.

White, black, blue and yellow irradiationsreduced the percentage of spore germination byabout 10 % compared to that of dark-growncultures, while red had no effect on spore germina-tion. Similar results have been reported byCochrane (1945) in that light of high intensities(1250 f-c) reduced germination of Phragmidiummucronatum on water agar to 45 %of the unillumi-nated controls. Lucas, Kendrick & Givan (1975)also found that cont inuous irradiation inhibitedurediniospore germination of Puccinia graminisf. sp, tritici.

The germ-tubes of V. agaricinum grow well,form hyphae and branch under dark as well asunder white, black, red, blue and yellow irradia-tion over a period of 22 h. In the case of near-u .v,

irradiation, the germ-tubes were short and theirdevelopment was very slow. This may explainthe whole appearance of the colonies grown undernear-u.v, irradiation when they appeared deeplyorange-coloured and wrinkled (Osman & Valadon,1979).

Previous studies (Osman & Valadon, 1978a, b,1979) and the present results show clearly thatnear-u.v. has a great effect on the morphology ofV. agaricinum especially as viewed under the lightmicroscope. An electron microscope study of suchaffected hyphae was therefore undertaken as it washoped that differences would be more obviousat the ultrastructure level.

The fungus was grown for 72 h in the dark orunder near-u.v. irradiation. The fungal hyphaewere chosen from the colony margins. Smallblocks of agar containing the fungal hyphae werecut and fixed in 1 % (vI v) glutaraldehyde insodium cacodylate buffer pH 7'2 for 30-60 min.The blocks were washed in buffer and post-fixed in1 % aqueous osmium tetroxide for 2 h. They werewashed in buffer again and placed in 0'5 % (w/v)aqueous uranyl acetate for 2 h. The agar blockswere then dehydrated in a graded ethanol/waterseries and in a mixture of ethanol/propylene oxide(1: 1, v/v) and finally in propylene oxide, and thenembedded in Spurr's resin (Spurr, 1969). Blockswere sectioned with a glass knife, stained withReynolds lead citrate «Pb(NOa)2 1'33 g; Na a-(C6Hs0 7.2H20 ) 1'76 g; 30 ml CO2-free H 20 ) andexamined using a Carl Zeiss ElektronenmikroskopeEM 9.

It is known from our previous studies that cul-tures exposed to near-u.v. irradiation contain largeamounts of carotenoids (Osman & Valadon,1978b ) and lipids (Osman & Valadon, 1980) com-pared to dark-grown cultures. So, as was expected,the most obvious ultrastructural changes were thepresence of large lipid globules. A further obvious

Trans. Br. mycol, Soc. 77 (1), (1981). Printed in Great Britain

Notes and brief articles

difference was the wall thickness of near-u.v.«grown hyphae (T S 0'148 ± 0'008, LS 0'144 ±0 '01 pm) which was almost twice that of dark-grownhyphae (TS 0'080 ±0'005, LS 0'078 ±0'007 pm).This result is in agreement with that obtained byVail & Lilly (1968) who found that the cell walls oflight-grown cells of Dacryopinax spathularia werebetween t- 5 and 1 '9 times as thick as those of dark-grown cells. Furthermore, in some of the near-u.v.-treated cultures of V. agaricinum the hyphaeappeared to be almost completely made up of lipidglobules.

In conclusion then, it appears that the near-u.v,part of the spectrum is most effective in decreasingthe percentage of spore germination, in increasingthe thickness of cell walls and in increasing thenumber and size of lipid globules as well as thecarotenoid content of V. agaricinum.

REFERENCES

BERG, L. A. & GALLEGLY, M. E. (1966). Effect of lighton oospore germination in species of Phytophthora.Phytopathology 56, 473-482 .

COCHRANE, V. W. (1945). The effect of artificial lighton germination of uredospores of Phragmidiummucronatum (Fr.) Schlecht. Phytopathology 35, 458-462.

HARDA, Y. (1975). Culture studies on the moniliadisease fungus of apples. VI. The effect of light onconidial germination, mycelial growth and sporu-lation. Bulletin of the Faculty of Agriculture ofHiro saki University 25, 33-39.

LUCAS, J. A., KENDRICK, R. E. & GIVAN, C. V. (1975).Photocontrol of fungal spore germination. PlantPhysiology 56, 847-849.

OSMAN, M. & VALADON, L. R. G. (1978a). Effect oflight quality on the photoinduction of carotenoidsynthesis in V erticillium agaricinum. Microbios 18,220-234.

OSMAN, M. & VALADON, L. R. G. (1978b). Studieson the near-uv effect on carotenogenesis in Verti-cillium agaricinum. Microbios 23, 53-64.

OSMAN, M. & VALADON, L. R. G. (1979). Effect oflight quality on growth and sporulation in Verti-cillium agaricinum, Transactions of the British Myco-logical Society 72, 146-147.

OSMAN, M. & VALADON, L. R. G. (1980). The effectoflight quality on the lipid composition of Verticilliumagaricinum. Microbios Letters 13, 59-73.

SCHNEIDER, M. J. & MURRAY, B. J. (1979). Phyto-chrome mediation of uredospore germination in thefungus Puccinia graminis . Photochemistry and Photo-biology 29, 1051-1052.

SPURR, A. R. (1969). A low-viscosity epoxy resinembedding medium for electron microscopy.Journal of Ultrastructure Research z6, 31-43.

VAIL, W. J. & LILLY, V. G. (1968). The location ofcarotenoid pigments and thickness of the cell wallin light- and dark-grown cells of Dacryopinaxspathularia. Mycologia 60, 902-907.

VALADON, L. R. G. & MUMMERY, R. S. (1971). Effectof light on nucleic acids, proteins and carotenoidsof Verticillium agaricinum. Microbios 4, 227-240.

VALADON, L. R. G., OSMAN, M. & MUMMERY, R. S.(1979). Phytochrome-mediated carotenoid synthesisin the fungus Verticillium agaricinum. Photochemistryand Photobiology 29, 605-601.

ZADOKS, J. C. & GROENEWEGEN, L. J. M. (1967). Onlight-sensitivity in germinating uredospores ofwheat brown rust. Netherlands Journal of PlantPathology 73, 83-102 .

EFFECT OF RUBRATOXIN B ON AMINO ACID COMPOSITIONOF THE HYPHAL WALL OF ASPERGILLUS NIGER

M. O. MOSS AND F. BADII

Department of Microbiology, University of Surrey,Guildford, Surrey GU2 5XH

The rubratoxins are nonadrides produced byPenicillium rubrum Stoll which show some antifungalactivity (Reiss, 1972). During a study of the effectsof rubratoxin B on the growth, metabolism andmorphology of Aspergillus niger Van T iegh.,significant changes were observed in the chemicalcomposition of hyphal wall preparations (M oss &Badii, 1980). These changes accompanied a reduc-tion of growth rate and dramatic alterations in themorphology and metabolism of the whole organ-

ism . One of the changes observed was a reductionin the protein content of wall material from approx-imately 11'0 % in normal mycelium to approx-imately 8'0 % in rubratoxin-treated mycelium.

It is now widely accepted that protein forms animportant constituent of the hyphal wall of fungi.Reported values of protein content vary from aslittle as 3-6 % in the walls of Leptomitis laeteus(Roth.) Agardh, (Aronson & Lin, 1978), 7'2 %in Penicillium rub rum Stoll (Unger & Hayes, 1975)

Tran s. Br, mycol. Soc. 77 (1), (1981). Printed in Great Britain