Notes and Brief Articles 309

A. crenulatus and that this might have contributed to the observed effects ofthe antagonist through the cellophane membrane.

The antagonism shown by P. gigantea against H. annosum in bothnatural and artificial situations is possibly effected by means of hyphalinterference.

We are indebted to Dr ]. Rishbeth for generously making available aculture of P. gigantea and for helpful discussion.

REFERE~CES

GIBBS, j. N. F. (1967a). A study of the epiphytic growth habit of Fornes annosus. Ann. Bot.N.S. 31, 755-744.

GIBBS,j. N. F. (1967b). The role of host vigour in the susceptibility of Pines to Fornesannosus. Ann. Bot. N.S. 31,803--815.

IKEDlUGWU, F. E. O. & WEBSTER,j. (1970a). Antagonism between Coprinus heptemerusand other coprophilous fungi. Trans. Br. mycol. Soc. 54, 181-204.

IKEDlUGWU, F. E. O. & WEBSTER,j. (1970b). Hyphal interference in a range of copro­philous fungi. Trans. Br. mycol. Soc. 54, 205-210.

MEREDITH, D. S. (1960). Further observations on fungi inhabiting Pine stumps. Ann.Bot. 24, 63-78.

RIsHBETH,j. (1950). Observations on the biology of Fornes annosus, with particularreference to East Anglian pine plantations. I. The outbreak of the disease and eco­logical status of the fungus. Ann. Bot. ~.S. :14, 365~-383'

RISHBETH,j. (1952). Control of Fomes annosus Fro Forestry 25, 41-5°.RISHBETH,j. (1961). Inoculation of pine stumps against infection by Fornes annosus,

Nature, Lond. 191, 826-827.RISHBETH,J. (1963). Stump ptotection against Fornes annosus. III. Inoculation with

Peniophora gigantea. Ann. appl. Biol. 52, 63-77.

EXPLANATION OF PLATE 26Hyphal interference by Peniophora gigantea, growing from left to right, against Heterobasidion

<2nnosum, growing upwards.Fig. I. The zone of contact between a colony of P. gigantea and one of H. annosum. The contactedcells of the Heterobasidion are killed (arrows) and are either transparent or granular.Fig. 2. Remains of the cell contents of the affected cells of H. annosum (arrows) are mainlylocated at the point of contact with the Peniophora hyphae.

ULTRASTRUCTURE OF AN ALBI~-o STRAINOF ALTERNARIA BRASSICICOLA

R. CAMPBELL

Department of Botany, The Unioersiiy, Bristol BS8 1 UG

The origin and general characteristics of an albino strain of Alternariahrassicicola (Schw.) Wiltshire, have already been published (Campbell,Lamer & Madelin, 1968): this report compares its ultrastructure withthat of the wild type (Campbell, 1968; 1969; 1970).

The albino strain was grown on potato-dextrose agar at 25°C for

Trans. Br. mycol. Soc. 54 (2), (1970). Printed in Great Britain

310 Transactions British Mycological Societystudies of the hyphae, conidiophores and spores. Germinating spores weretaken from a glucose-asparagine liquid medium with forced aeration. Theformation of melanin-like pigments was induced by growing the albino invigorously aerated glucose-asparagine liquid medium saturated with tyro­sine. Cytochemical tests were based on those of Pearse (1953), Jensen(1962) and Breslau (1962). Several methods of preparation for electronmicroscopy were used (see Campbell, 1969 for details): material was fixedin 2 % (wjv) unbuffered potassium permanganate, dehydrated in analcohol series and embedded in epon. Freeze-etching was performed byMoor & Muhlethaler's (1963) method with a Balzer's freeze-etching unit.Preparations of wall material, after cleansing in saturated potassiumhydroxide, were shadowed with platinum-carbon.

The structure of the hyphae and the conidiophores was very similar tothat of the wild-type (Campbell, 1970) except that there were no electron­dense deposits of melanin in the wall and the pore in the conidiophore tiphad no annulus (Campbell, 1968). Spores were produced normally, i.e. bythe extrusion of the inner wall layer through a pore in the outer layer.

The cytoplasm of the conidia often contained large amounts of anelectron-transparent substance (PI. 27, fig. I), which also occurred in muchsmaller quantities in the wild type. It has been identified cytochemicallyas a partially sulphated polyglucoside. All the normal organelles and alsolipid bodies were present in the conidia (PI. 27, fig. I; PI. 28, fig. 5), but theplasmalemma lacked the grooves in its outer surface and correspondingridges on the inner one (PI. 27, fig. 2), which were so common in thewild type.

Septal partitions in the albino conidia had the same three-layeredstructure (PI. 27, figs. I, 3) as in the wild type, and both primary andsecondary conidial walls were present (PI. 27, figs. 1,3) though the layerswere not always clearly defined because of the lack of pigmentation. Onthe outside of the conidial wall there was a unit membrane (PI. 27, fig. 3),usually with a layer of amorphous material beneath. The origin andfunction of this membrane are unknown. The walls were composed offibrils (PI. 27, fig. 4) which cytochemical tests suggested were chitin em­bedded in a matrix of acidic polysaccharide(s). As the primary wall agedit sometimes became very tenuous (PI. 28, fig. 5) presumably becausemelanin was not deposited. In aqueous and particularly strongly ionicsolutions, the fragile, unmelanized primary wall and septal partitionsusually disintegrated so that the spore broke apart into its constituent cells(Campbell et at. 1968) each surrounded by its own secondary wall(PI. 28, fig. 5). The collapse of the tenuous primary wall, and the wrinklingof the overlying membrane, accounts for the rough appearance of thespores, particularly after drying (Campbell et at. 1968).

Germination was affected by the wall structure. Intact spores ger­minated in the normal way, by the growth of the secondary wall througha break in the primary one (PI. 28, fig. 6). If, however, the spore dis­integrated into its constituent cells each of these germinated by extensionof the secondary wall which was then the only envelope (PI. 28, fig. 7). The

Trans. Br. mycol. Soc. 54 (2), (1970). Printed in Great Britain

Notes and Brief Articles 311germ tube was frequently constricted at the base even though it did nothave to pass through the primary wall.

When spores of the albino were produced in submerged liquid culturewith excess tyrosine they developed normally. They were true porosporesand had an electron-dense, brown substance, presumably very similar tomelanin, in their primary walls and septal partitions (PI. 28, fig. 8). Theirplasmalemmas had grooves in their outer surfaces (PI. 28, fig. 9) like thosepresent in wild-type spores. They did not disintegrate in aqueous ionicsolutions. No annulus was seen in the treated albino.

All the structural differences between the wild and albino strains canbe accounted for by the absence of melanin from the latter. Such differ­ences as the increased amounts of polyglucoside could be caused by minorshifts in metabolic equilibria that result from the failure to synthesizemelanin.

Various functions have been assigned to melanin in fungi. It may pro­tect the spore from radiation damage and from enzymic attack from othermicro-organisms during dormancy in the soil (Kuo & Alexander, 1967and their co-workers). These roles have been demonstrated in A. brassici­cola (author's unpublished results). A structural role has been proposedfor melanin (Bu'lock, 1967) but little evidence was presented. Thedisintegration of albino spores in ionic solutions when melanin is absentindicates that the structural function may be important. A very large three­dimensional polymer such as melanin is well suited to structural purposesespecially when it occurs in such large amounts as in the walls of thewild-type. Apart from this physical aspect the melanin could also formhydrogen bonds with the wall polysaccharides, increasing their stabilityand preventing their dissociation in ionic solutions, such as zinc-chlor­iodide (Campbell et al. 1968). The stability of the spores of the albinostrain with tyrosine-induced pigmentation supports these arguments forthe structural role of melanin.

Tyrosine was used as a precursor in these experiments because of its lowtoxicity and high chemical stability. Since the albino produced brownmelanin-like pigments if provided with tyrosine it must possess the enzymetyrosinase. Preliminary comparisons of the activity of tyrosinase in thewild and albino strains indicated little, if any, difference. Tyrosine is aconstituent of many enzymes and structural proteins; it is unlikely to belacking in the albino which had very similar rates of growth and sporula­tion to those of the wild-type. Thus the mutation is probably not in themelanin synthesis pathway itself and its most likely location is in thetransport mechanism across the plasmalemma. Probably tyrosine pro­duced in the cytoplasm could not be transported to the primary wall andseptal partitions which is where the formation of pigment from exo­genously supplied tyrosine indicates that the tyrosinase is located.

The grooves in the outer surface of the plasmalemma of the spores occurin the wild-type and in the albino with exogenous tyrosine but not in theuntreated albino. The grooves may therefore be connected with melaninsynthesis but not with the tyrosine transport.

Trans. Br. mycol. Soc. 54 (2), (1970). Printed in Great Britain

312 Transactions British Mycological SocietyLuttrell (1963) in studies on Helminthosporium, distinguished two types of

conidia, euseptate and distoseptate, and suggested that the distinctionmight be useful in taxonomy. The wild-type spores of Alternaria brassicicolaare apparently euseptate in that they maintain their integrity as multi­celled spores, even though each cell is surrounded by its own wall (Camp­bell, 1968). The albino spores are distoseptate. The distinction betweenthe two types is difficult at the ultrastructural level and since they bothoccur in strains of a single species their usefulness as taxonomic criteria isdoubtful, though they may be of value in determinative work.

Modern classification in the Deuteromycetes (based on Hughes, 1953)depends largely on the ontogeny of conidia. The wild-type of A. brassicicolais a porospore and so is the albino even though it lacks an annulus and itswall structure is somewhat different. All known porospores have mela­nized, often thick, walls, a feature which may have necessitated thedevelopment of porogenous spore production. This study therefore pro­vides evidence that porogeny is a sufficiently stable character to persistdespite changes in wall structure, and is, therefore, a valid taxonomiccriterion.

I wish to thank Professor L. E. Hawker, Dr M. F. Madelin and Dr A.Beckett for help and advice throughout this work which formed part of aPh.D. thesis presented in the University of Bristol.

REFERENCES

BRESLAU, A. M. (1962). Polysaccharides in Micro-organisms. In Handbuch der Histo­chemie (ed. by W. Graumann & K. Neumann). Bd. 2. Teil I, pp 1-94. Stuttgart:Fischer Verlag.

BU'LOCK,J. D. (1967). Essays in biosynthesis and microbial development. New York: JohnWiley and Sons Inc.

CAMPBELL, R. (1968). An electron microscope study of spore structure and developmentin Alternaria brassicicola. J. gen. Microbiol. 54, 381-392.

CAMPBELL, R. (1969). Further electron microscope studies of the conidium of Alternariabrassicicola. Arch. Mikrobiol. 69, 60-68.

CAMPBELL, R. (1970). An electron microscope study of exogenously dormant spores,spore germination, hyphae and conidiophores of Alternaria brassicicola. New Phytol.69, 287-294.

CAMPBELL, R., LARNER, R. W. & MADELIN, M. F. (1968). Notes on an albino mutant ofAlternaria brassicicola. Mycologia 60, 1122-1125.

HUGHES, S. J. (1953)· Conidiophores, conidia and classification. Can.J. Bot. 31 , 577-659.JENSEN, W. A. (1962). Botanical histochemistry. San Francisco and London: W. H.

Freeman and Co.LUTTRELL, E. S. (1963). Taxonomic criteria in Helminihosporium. Mycologia 55,643-674.Kuo, M.J. & ALEXANDER, M. (1967). Inhibition oflysis of fungi by melanins. J. Bact.

94, 624-629.MOOR, H. & MUHLETHALER, K. (1963)' Fine structure in frozen-etched yeast cells.

J. Cell Biol. 17, 609-628.PEARSE, A. G. E. (1953). Histochemistry, theoretical and applied. London: J. and A. Chur­

chill, Ltd.

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Notes and Brief Articles

EXPLANATION OF PLAT ES 27 AN D 28Albino Alternaria brassicicola

P LATE 27

Fig. I. A mature spore with a la rge amount of partially sulphated polyglucoside (PG) and alsothe no rmal organelles. T he primar y (P W) and the secondary (SW ) walls are show n. K Mn04•

x 10 0 0 0 .

Fig. 2 . T he inner surface of the plasm alemma of a spore ; note the abs ence of ridges whic h a re socommon in the wild- typ e. Freeze-e tched ; the a rrow ind icates the d irec tion of the shadowing.x 30000.

Fig . 3. The wall of a mature spore ; the p rimary (PW) and secondary wa lls (SW) , septal par­tition (SP), wa ll membrane ( WM) and the underlying amorp hous layer (a t arrow) a re shown .KMn04 • x 37500.

Fig. 4. The fibril s, probably of chi tin, of which the wall is composed. Chemically cleaned andshadowed with pl ati num-car bon . x 75000.

PLATE 28

Fig. 5. A spore in wh ich th e primary wall has completely disint egrated , the cells of tb e sporeenclosed in their secondary walls (SW) a rc about to separa te as the wa ll membrane (W.A;f)breaks. KMnO•. x 6000.

Fig. 6. Germination of an in tact spore with th e secondary wall (SW) breaking through th eprimary wa ll (PW) in th e nor mal way . KMnO•. x 12 0 0 0 .

Fig. 7. A cell, from a disin tegrat ed spore, germinat ing by the extension of the one remainingwall, the seconda ry wa ll (SW ). Note the constriction at the base of th e ger m-tube, even thoughit does no t have to pass th rou gh a brea k in the: spore p rimary wall. K MnO•. x 3800.

Fig. 8. A part of a spore of the alb ino strain tha t has had pigm entation induced by tyro sine. Thepigment is in th e pr imar y wall (P W ) and to a lesser exte nt in the septa l partition (SP). K MnO•.x 10 0 00.

Fig. 9. The outer surface of the plasmalemma of an a lbino spore with induced pigmen ta tion;note the charac teristic grooves in th e surface which are a feature of th e wild-type rather th an thealbino spores (compare with Fig. 2). Freeze-etc hed; the arrow indicat es th e direction of theshad owing. x 22500.

THE FINE STRUCTURE OF BLASTOCr STISFROM THE CAECUM OF TURKEY

D. L.LEE

Department of Parasitology, Houghton Poultry Research Station,Houghton, Huntingdon, England

In the course of our work on the fine structure of the protozoon Histo­monas meleagridis in the tissues of turkeys (Lee, Long, Millard & Bradley,1969) we also came across sections of a fungus-like organism which welater showed, in cultures and in smears, to be Blastocystis. Because it isunusual in possessing an investing coat of 'mucilage ' and not a typicalcell wall, and as we can find no pu blished work on the fine structure ofthis fungus, it was decided to publish the results. This paper should alsohelp to clarify the role of Blastocystis in blackhead disease of turkeys.

Blackhead disease (entero-hepatitis) of turkeys is genera lly believed tobe caused by Histomonas meleagridis, but some authors claim th at this

Trans. Br. mycol. Soc. 54 ( 2 ) , ( 19 70) . Printed in Great Britain