Arch. Mikrobiol. 64, "279--288 (1969)

The Uhrastructure of the Stroma of the Brown Rot Fungi

H. J. WILLETTS and F. D. CALONG]~

Department of Botany, University of Bristol

l~eceived August 19, 1968

Summary. Most of the hyphae forming the medulla of the stroma of the brown rot fungi are 4--7 tz in diameter and contain food reserves in large vacuoles and lipid bodies. Some stromatal hyphae have very thick walls and perform a protective function. Smaller hyphae (1--2 ~ in diameter) form a network through the medulla and their structure suggests that they initiate the growth of vegetative hyphae and spores after the stroma has passed through a period of rest.

In fo rmat ion on the s t ructure of s t romata l and sclerotial hyphae is l imited. GO,DEE and PORTER (1961) reported t h a t microsclerotia of Verticillium albo-atrum are composed of thick and thin-walled cells and tha t only the thin-walled cells germinate. I n u l t ras t ruc tura l studies of the same fungus NADAKAVUKA~N (1963) also found tha t the micro- sclerotia are composed of thick and thin-walled cells in close association and tha t the larger, thick-walled cells conta in food vacuoles and p robab ly serve the dual funct ions of protect ion and food reserve.

The work described in this paper presents data on the u l t ras t ruc ture of the s t roma of the brown rot fungi [viz. Sclerotinia ]ructicola (~Vint.) l~ehm, S. ]ructigena Aderh. et RUM., S. laxa Aderh. et l~uhl, and S. laxa forma mali (Wormald) Harrison] and forms par t of a general inves t igat ion of these fungi.

Methods Transmission electron microscope studies were made of mature stromata of the

European brown rot fungf (viz. all the species apart from S./ructicola) grown on malt or potato dextrose agar and in artificially infected fruits. The materials were fixed either in 2 per cent unbuffered potassium permanganate or in 6 per cent glutaraldehyde followed by 2 per cent osmium tetroxidc, embedded in Epon, sectioned by methods previously described (WILL~TTS and C~O~G~, 1969) and examined and photographed with an A.E.I. EM 6B Electron Microscope operating at a potential of approximately 60 k.V.

Parallel material was fixed in F.A.A. and sections, 5--10 tz thick, were cut with a freezing microtome. These were stained with cotton blue in lactophenol, mounted in lactophenol and viewed with a light microscope.

20 Arch. 3iikrobiol., Bd. 64

280 H.J. WILLETTS and F. D. CALONGE:

Figs. l--4. Electron micrographs of transverse sections of stromatal hyphae. Material fixed in glutaraldehyde and osmium tetroxide

Fig. 1. A storage hypha with a large vacuole (V), one obvious lipid body (L), un- identified bodies (B) between the plasmalemma (P1) and the cell wall (W), and

poorly defined organelles (0). • 37,800

Results

General Structure of Stroma

The s t roma of the brown rot fungi consists of a medulla of closely interwoven hyphae which is covered on all exposed surfaces by a black or dark brown rind, several cells thick. A description of the s tructure of the r ind has been given in an earlier paper (WILL~TTS, 1968). The medulla consists mainly of closely interwoven hyphae that , individually, are similar in size and appearance to ordinary vegetat ive hyphae. Medullary hyphae sometimes have regular constrictions and have the

Stromatal Ultrastructure 281

Fig.2. A storage hypha with a large vacuole (V), nmnerous lipid bodies (L) and a cell wall (W) in which layering can be seen. X 45,500

same appearance as chains of maeroeonidia in which septa have not developed. When thin sections of the s troma are stained with cot ton blue in laetophenol, the hyphae tha t form the main par t of the medulla do not take up the stain but a network of fine, thin-walled, deeply staining hyphae becomes apparent. Even in old s t romata these hyphae stain deeply.

Ultrastructure of the Large Stromatal t Iyphae

Most of the hyphae in the interior of the s t roma are about 4 - - 7 in diameter, with their walls approximate ly 0.25 a thick (Figs. 1 and 2). Occasionally, wMls up to 1.8 ~ thick were observed and in them in- dist inct concentric layers were sometimes discernible (Fig. 3). Such cells

20*

282 tI. J. WrLLETTS and F. D. CA~O~G~ :

Fig. 3. A hypha with a thick wall (W) and small amounts of food reserves. • 15,940

usually showed signs of cell autolysis. The walls were usually made up of a single layer although some hyphae had walls with two distinct regions, a thin (0.04 ~) electron dense outer layer and an in inner layer approx. 0.22 ~ thick (Fig.4). The appearance of the walls of these hyphae is similar to that of aerial vegetative hyphae and young conidio- phores (WmLErrS and CALO~GE, 1969).

In most cells the plasmalemma was undulating and clearly defined but in cells with thick walls or in those containing large amounts of food reserves, the plasmalemma was sometimes incomplete. Fig.5 shows a perforated plasmalemma and just outside it are numerous structures which vary in shape and size. Some are tubular, others are vesicular and they are similar in appearance to the lomasomes first described by Moo~E and McALEA~ (1961). Larger but similar vesicles can be seen

Stromatal Ultrastructure 283

Fig.4. Note the wall which has an outer electron dense layer (OW). • 16,170

within the protoplast. The "lomasomes" were seen only in material fixed with potassium permanganate.

Mature stromatal cells contain fewer organelles than do the clearly- structured protoplasts of young vegetative cells and the membrane- contrast of the organelles that remain is partly lost.. The main inclusions in these cells are several small vacuoles, or a single large one, which may occupy the greater part of the cell, and lipid bodies which become fewer but larger as the stroma ages (Figs. 1 and 2). The vacuoles contain numerous small electron-dense particles suggesting that they are prob- ably rich in reserve food materials. Where extreme thickening of a cell had taken place the vacuoles and lipid bodies were few and small. The food reserves were probably used up during the deposition of materials on the walls. The cytoplasmic continuity between neighbouring cells

284 H. J . WrLr,]~TTS and F. D. CALONGE

Fig. 5. Transverse section of stromatal hypha fixed in potassium permanganate and stained in uranyl acetate. Note the open nucleus (N), poorly defined organelles, an incomplete plasmalemma (P1) with numerous small electron dense areas (T)

outside it, and a cell wall (W) made up of several layers. • 41,400

along a h y p h a is ma in t a ined b y a s imple pore b u t as the s t roma ma tu re s the pores are b locked or t hey are sealed b y fur ther g rowth of the septum. I n longi tud ina l sections, constr ic t ions are somet imes seen along the length of a h y p h a and the d is tance be tween constr ic t ions is a b o u t 10 ~ (Fig. 6). The contraction of the protoplast shown in Fig. 6 may have been caused by the techniques or solutions used to fix the material but the unidenti- fied structures in the space between the plasmalemma and wall are likely to be of some significance in the contraction of the protoplast.

Ultrastrlleture of the Fine Hyphae of the qiV[edulla

The hyphae that form a fine network through the stroma and stain deeply with cotton blue in lactophenol have a diameter of about 1--2 bt.

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286 H.J. WrLL~TTS and F. D. CALONGN:

Fig. 7. Note the plugged pore (Pr), septum (S), electron dense areas (T) outside the plasmalemma (P1), mitochondria (Mr). • 31,050. Fixed in potassium permanga-

nate and stained in uranyl acetate

They have walls that are made up of a single, slightly electron dense layer approx. 0.12 ~ thick and they contain numerous clearly defined organelles (Figs. 7 and 8). These include several nuclei, a system of membranes in the endoplasm and round or globose mitoehondria. Lipid bodies and vacuoles are not usually present in these hyphae. Small tubules and vesicles, the same as those observed in the larger, non-staining hyphae of the medulla, were present outside tile plasma- lemmae.

Discussion

Some stromatal hyphae have very thick walls and probably they are found in greatest numbers in the exposed parts of the stroma where they have a protective function. The rounded cells seen with a Stereoscan electron microscope in gaps in the crust that covers the surface of stromata (WILLETTS, 1968) would be expected to have this structure.

Stromatal Ultrastructure 287

Fig. 8. Note the small diameter of this hypha, the clearly defined organelles and the electron dense areas (T) outside the plasmalemma (PI). • 20,440. Fixed in

potassium permanganate and stained in uranyl acetate

The majori ty of medullary cells are rich in food reserves and their organelles are poorly defined and few in number. Although the main function of such cells is food storage under certain conditions they may retain the ability to undergo further growth.

The well-defined organelles of the small hyphae tha t form a network in the stroma suggest tha t these cells are metabolically active and initiate the growth of vegetative hyphae and spores after the stroma has passed through a period of rest. The small hyphae are in close contact with the larger ones tha t contain abundant food reserves and it is prob- ably from these tha t they obtain the energy for further growth.

The small tubules and vesicles outside the plasmalemma of some cells are probably similar to the lomasomes first described by Moo~E and McALEA~ (1961) and observed in a variety of fungi since that t ime (see reviews of HAWKER, 1965; MOORE, 1965). BnACKEr~ (1966) in a study of

288 H. J . W r n r ~ s and F. D. CA~O~CE : StromatM Ultrastructure

sporangiospore wall f o rma t ion b y GilberteIla persicaria descr ibed the deve lopmen t of e lect ron dense envelopes outs ide the p l a sma le mma e of young spores. The s t ruc tures observed in the presen t inves t iga t ion are similar to the granules r epo r t ed b y B~)~cKE~ and are p r o b a b l y associa ted with the th ickening of the h y p h a l walls. The presence, wi th in the pro to- p las t of a cell undergoing autolysis , of small vesicles s imilar to those found outs ide p l a sma lemmae suggests t h a t these vesicles are formed inside the p ro top l a s t and cont r ibu te to wall th ickening. The concentr ic layer ing observed in the walls of some s t roma ta l h y p h a e m a y be f rom th ickening of th is type .

A th in e lectron-dense layer was found on the outer surface of macro- conidia and aerial h y p h a e of the brown ro t fungi (W~L~TTS and CALo~G~, 1969) and i t was suggested t h a t i t is formed b y the collapse and dry ing out of an outer, p r o b a b l y muci laginous layer . The absence of an e lect ron dense layer on the outer surface of most of the h y p h a e in m a t u r e s t r o m a t a m a y indica te t h a t only the hyphae on the exposed surfaces of the s t roma are sub jec ted to dry ing condit ions. Obvious ly the compac t organisa t ion of the medu l l a ry h y p h a e will reduce des iccat ion of the inner cells b u t the r ind of th ickened, closely-fi t t ing cells mus t also help to p reven t the loss of mois ture f rom the s t roma.

A e kn o wle dg m e n t s. The authors are indebted to Professor L. E. H ~ w K ~ and Dr. J. R. W. BY,DE for their encouragement and help during this investigation and to Mr. HA~s Cook for photographic assistance. Tha.nks ~re due to the Agricultural Research Coucil for ~ grant to one of us (F.D.C).

References B~ACKER, C. E. : Ultrastructural aspects of sporangiospore formation in Gilbertella

persicaria. In: The ]ungus spore (ed. M. F. MAD~LI~), pp. 39--59. London: Butterworths 1966.

GORDEE, R. S., and C.L. PoRTER: Structure, germination and physiology of microselerotia of Verticillium alboatrum. Mycologia, 53, 171--182 (1961).

H~WKER, L. E. : Fine structure of fungi as revealed by electron microscopy. Biol. Rev. 40, 52--92 (1965).

MooR~, R. T. : Th~ ultrastructure of funga] cells. In The fungi (ed. G. C. AI~SWORT~ and A. S. Suss~A~) pp. 95--118. New York: Academic Press 1965.

- - , and J .H . McALEAn: Fine structure of mycota. 5. Lomasomes-previously uncharacterized hyphal structures. Mycologia, 53, 194--200 (1961).

NADAKAVUKAm~, M. J. : Fine structure of microsclerotia of Verticillium albo- atrum l~einke et Berth. Can. J. Microbiol. 9, 411--413 (1963).

W I L L E T T S , I-~. J. : Stromatal rind formation in the brown rot fungi. J. gem Microbiol. ~2, 271--273 (1968).

-- , and F. D. CALONG]~: Spore development in the brown rot fungi (Sclerotinia spp.). New Phytol. (In press, 1969).

Dr. l-I. J. WILLETTS l)r. F. D. CaLO~GE School of Botany, University of Instituto Botanieo "A. J, Cavanilles" New South Wales, (C.S.I.C.) Kensington, N.S.W., Australia Madrid, Spain