Anatomy and histochemistry of resting and germinating sclerotia of Sclerotium cepivorum

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Primed in Great Britain

ANATOMY AND HISTOCHEMISTRY OF RESTING ANDGERMINATING SCLEROTIA OF SCLEROTIUM CEPIVORUM

By D. BACKHOUSE AND A. STEWARTBotany Department, University of Auckland, Auckland, New Zealand

Sclerotia of Sclerotium cepivorum had a rind one or two cells thick, no cortex, and a medullain which all interhyphal spaces were filled with a polysaccharide gel. The main reservematerials were protein bodies in medullary hyphae. Sclerotia recovered from soil after severalyears burial showed extensive loss of hyphal contents although their rinds remained intactand many hyphae appeared healthy. During germination renewed hyphal growth in theoutermost parts of the medulla led to bulging and rupture of the rind followed by radiationof hyphae from a germinative plug.

Sclerotium cepivorum Berk . causes white rot ofAllium species. Its propagules are sclerotia, whichare relatively small (200-500 pm diam) but areproduced in large numbers on infected host tissue.Although significant decay of sclerotia may occurin a single season in organic soils in certain areas(Leggett, Rahe & Utkhede, 1983), in other soilssclerotia can survive for over 10 years with littleloss of viability (Coley-Smith , 1979).

Sclerotia of S. cepivorum consist of a narrowrind, whose cells have thickened pigmented walls,surrounding a medulla of closely interwoven, smalldiameter, moderately thick-walled hyphae withinterhyphal spaces filled with a gelatinous material(Mordue, 1976; Entwistle & Munasinghe, 1981;Georgy & Coley-Smith, 1982; Leggett & Rahe,1985). Mordue (1976) also described a cortex of2-3 layers of isodiametric thin-walled cells betweenthe rind and medulla.

In the field sclerotia germinate only in thepresence of susceptible hosts . Germination isstimulated by the breakdown products of flavourprecursors produced by Allium species but thebiochemical nature of this effect is little understood(Esler & Coley-Smith, 1983). In soil sclerotiausually germinate by producing an eruptive hyphalplug (Coley-Smith , 1960). Under axenic conditionssurface sterilized sclerotia may germinate erupti-vely but can also germinate by producing manyindividual hyphae (Coley-Smith et al., 1967).

In this paper we describe in more detail theanatomy of resting and germinating sclerotia of S.cepivorum, the identity and distribution of reservematerials, and changes to sclerotia during germi-nation and prolonged periods of burial.

MATERIALS AND METHODS

Sclerotia were extracted from soil from a field atPukekohe, South Auckland, New Zealand that hadbeen free of Allium species for three years, usingthe wet sieving-flotation technique of Crowe et al.(1980). An onion crop sown in this field aftersampling showed high levels of disease. Sclerotiawere also collected from diseased onions fromPukekohe, and an isolation made from these wasused for culture. Cultured sclerotia were producedon potato dextrose agar (PD A) in 9 em Petri dishesmaintained at room temperature for 4 wk, or on amedium containing horticultural pumice (500 g),fine maizemeal (25 g) and deionized water (100 ml)in 2 I flasks incubated at room temperature for 6wk. Sclerotia from pumice-maizemeal cultureswere stored for 6 wk at 10 °C before examination.To study germination, 6-wk-old sclerotia weresurface sterilized with I % sodium hypochlorite for10 min, plated onto PDA and harvested after 2 and4 d incubation at room temperature.

Sclerotia from each source were cut in half andfixed in 3 % glutaraldehyde in 0· I M phosphatebuffer, pH TO, at 4° for 4 h, then dehydrated inethanol and embedded in glycol methacrylate(G MA ; Feder & O'Brien, 1968). Sections 1-2 pmthick were cut on an ultramicrotome and used forlight microscopy. For transmission electron micro-scopy (T EM) sclerotia from PDA cultures werefixed as above, then post-fixed in I % osmiumtetroxide in 0·1 M phosphate buffer, pH 7.0, for2 h at 4°, dehydrated in ethanol and propyleneoxide and embedded in Agar 100 resin (Agar AidsLtd, Stansted, Essex ). Thin sections were stainedwith uranyl acetate followed by lead citrate.

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Staining procedures

All staining of GMA sections was done at roomtemperature. Stained sections were rinsed, air-dried and mounted in immersion oil.

Acid fuchsin . Sections were stained for 10 minwith I % aqueous acid fuchsin. Basic substances,including protein, stain red (Feder & O'Brien,1968) .

Coomassie blue. Sections were stained for 30 minwith 0'25 % aqueous Coomassie brilliant blueGZ50, then destained with I: 2: 7 acetic acid :meth-anol: water. Proteins stain blue (Cawood, Potter& Dickinson, 1978).

Fast blue B. Sections were stained for 5 min with0'2 % Fast blue B in 0'1 M Tris-HCI buffer, pH9'0, rinsed, then coupled for 5 min with I %resorcinol in the same buffer. Proteins and somephenolic materials stain red (Pearse, 1985).

Toluidine blue. Sections were stained with 0'5 %toluidine blue in 0'1 M acetate buffer, pH 4'4, for15 min. Acidic groups stain shades of blue to red,and phenolics green (Feder & O'Brien, 1968). Tocontrol ionization of tissue substances, some sec-tions were stained for 30 min with 0' I % toluidineblue acidified to pH 1'0 with hydrochloric acid.Polyphosphates, polysulphates and some polycar-boxylic acids stain red (Ashford, Ling Lee &Chilvers, 1975).

Lead sulphide. Sections were stained with 10 %lead nitrate in 0'1 M acetate buffer, pH 4"5, for15 min, rinsed, then treated with 10 % aqueoussodium sulphide for 10 min. Polyphosphates stainblack (Gahan , 1984).

Periodic acid-Schiff (PA S ) reaction. Sectionswere stained by the PAS procedure, followingaldehyde blockade with 2,4-dinitrophenylhydra-zine, according to the method of Feder & O'Brien(1968). Carbohydrates with free hydroxyl groupson adjacent carbon atoms stain red (Pearse,1985).

Photine H V . Sections were stained for 10 minwith 0'1 % aqueous Photine HV, air-dried withoutrinsing, mounted in oil and examined with a Zeissepifluorescence microscope using UGI, FT420

and LP4 I 8 filters. Cell walls and extracellularpolysaccharides fluoresce (Tampion, McKendrick& Holt, 1973).

Sudan black B . Sclerotia were encased in 20 %gelatin, hardened overnight with 5 % glutaralde-hyde, then sections cut by hand with a razor blade,stained for 10 min with saturated Sudan black B in70 % ethanol, and mounted in glycerol. SomeGMA sections were stained similarly. Lipids stainshades of blue or black (Pearse, 1985).

RESUL TS

Fresh sclerotia

Sclerotia from PDA and maizemeal cultures andfrom field-infected onions were very similar (F ig.I ). The rind was one cell, or occasionally two cells ,thick. Rind cells were isodiametric, thick-walled,and all contained cytoplasm. The outer surface ofthe rind and any spaces between rind cells werecovered with a thick deposit of pigment. Medullaryhyphae were thinner-walled, with elongated com-partments. They were loosely arranged in a matrixof extracellular material. There was no corticallayer between the rind and medulla. No fragmentsof host tissue were seen within sclerotia frominfected onions or from soil.

Most medullary hyphae contained electron-dense bodies about 0'5-3/tm diam, which oftenoccupied over half of the cell profile (F ig. 2). InGMA sections these bodies stained with acidfuchsin, Coomassie blue and Fast blue B (Fig. I).They showed strong metachromatic staining withtoluidine blue at pH 4'4 (F ig. 3) but were unstainedat pH 1'0. Staining with anionic and cationic dyeswas weak in the hyphae closest to the rind. It wasconcluded that the bodies contained protein with ahigh proportion of acidic residues. No lipid orcarbohydrate could be detected in protein bodiesby Sudan black B or PAS staining.

In TEM sections rind cells contained osmio-philic membrane-bounded stru ctures (F ig. 4).These were generally larger than protein bodiesand filled most of the cell volume. They appeared

Figs 1-5. Sections of sclerotia from culture.Fig . I. Light micrograph stained with Fast blue B, showing narrow rind and loosely arranged medullaryhyphae with protein bodies (Pb).Fig . 2 . TEM of medulla cell containing protein bodies (Pb) and with extracellular material (E) surroundingthe wall (W) .Fig . 3. LM stained with tolu idine blue showing protein bodies (Pb) and polyphosphate granules (Pp),Fig . 4. TEM of rind. Rind cells cont ain vacuoles (V) with osmiophilic content s and are surrounded bypigment (Pi).F ig. 5. LM stained with PAS . Walls and extr acellular material (E) stain for carbohydrate.Fig. 6. Section of sclerotium from diseased onion, stained with Photine HV and viewed with fluor escencemicroscopy . Walls and extra cellular materia l fluoresce .

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to be vacuoles but their contents could not beidentified histochemically in GMA sections.

When GMA sections were stained with toluidineblue at pH 4"4 or pH 1'0, small metachromaticgranules were seen in some medullary cells,especially those close to the rind (Fig. 3). Thegranules also stained with lead sulphide and wereconsidered to be polyphosphate. Polyphosphategranules were never abundant and were notidentified in TEM sections.

Hand sections stained with Sudan black Bshowed faint staining, presumably of membranes,in medullary hyphae. No accumulations of lipidwere seen and no staining was observed in GMAsections.

The hyphal contents were generally poorlystained with the PAS procedure (Fig. 5). Somestrongly PAS-positive areas were seen in somehyphae but they were of very limited extent. Thissuggests that intracellular polysaccharides are notmajor reserves in sclerotia of S. cepivorum.

All interhyphal spaces within the sclerotiumwere filled with extracellular matrix. In TEMsections this appeared to be fibrillar (Fig. 2). InGMA sections both walls and matrix fluorescedwith Photine HV (Fig. 6) and stained with the PASprocedure (Fig. 5), although PAS staining of thematrix was relatively faint. No staining of thematrix was detected with the other dyes used. Thisindicates that the matrix consists of a neutralpolysaccharide, with other materials, if present,only forming minor components.

Sclerotia from field soil

Most sclerotia extracted from field soil were intact,with uninterrupted rinds (Fig. 7). Around theoutside of the rind empty cell profiles and collapsedhyphae were often seen (Fig. 8). There was alwaysat least one layer of cells with both pigmented wallsand cytoplasmic contents.

Many medullary hyphae had lost all contents,although their profiles remained rounded (Fig. 7).Their walls stained with toluidine blue andCoomassie blue, but PAS staining was weak,indicating that the walls had been significantlyextracted. The matrix surrounding these cells

appeared unaltered apart from faint staining withCoomassie blue and toluidine blue. The proportionof empty hyphal profiles was greatest in the centreof sclerotia. The remaining medullary hyphaecontained protein bodies (Fig. 8) and appearedhealthy. No signs of bacteria or parasitic fungiwere seen in these sclerotia.

Occasional sclerotia from field soil appeared tobe dead. The medullary hyphae had irregularoutlines, cytoplasmic definition was poor, andbacteria were present within the sclerotia(Fig. 9).

Germinating sclerotia

Germination could first be recognized in wholesclerotia after two days incubation by bulging ofthe surface. In section the bulges were associatedwith closely packed, strongly stained hyphaeimmediately beneath the rind (Fig. 10). Occa-sionally germinative hyphae pushed through be-tween the rind cells (Fig. 10). Germination moreusually occurred by formation of a plug ofmycelium which ruptured and displaced the rind(Fig. I I). After four days incubation sclerotia werecovered by up to four tufts of mycelium, seen insection as hyphae radiating from points of germi-nation (Fig. 12). In all cases the growth ofgerminative hyphae occurred from the outermostpart of the medulla. No primordia or organizingregions were seen.

During the initial stages of germination nochanges to medullary hyphae could be detected. Atthe later stage sampled, protein bodies had disap-peared from many hyphae, especially near the baseof growing hyphal plugs (Fig. 12). The hyphalwalls and extracellular matrix appeared to remainunaltered at this stage.

DISCUSSION

The main cytoplasmic materials found that couldfunction as reserves were protein bodies. Protein isnow known to be a major constituent of sclerotia ofmany fungal species (lnsell et al., 1985). Proteinbodies have also been identified histochemicallyand ultrastructurally in sclerotia of Sclerotinia and

Figs 7--9. Sections of sclerotia recovered from field soil.Fig. 7. Coomassie blue stain. Many hyphae have lost their contents and their walls stain.Fig. 8. Toluidine blue stain. Remaining hyphae contain protein bodies (Pb), Collapsed hyphal profiles arevisible on the outer surface of the rind.Fig. 9. Apparently dead sclerotium containing bacteria (arrows). Toluidine blue stain.Figs 10-12. Median sections through points of germination.Fig. 10. Individual hyphae emerging through rind from bulge on sclerotial surface. Coomassie blue stain.Fig. I I. Hyphal plug emerging through ruptured rind. PAS stain.Fig. 12. Hyphae radiating from eruptive plug. Medulla hyphae near base of plug show reduced staining.Coomassie blue stain.

566 Sclerotium structure in Sclerotium

Botrytis species (Saito, 1977; Bullock, Ashford &Willetts, 1980; Backhouse & Willetts, 1984). Theprotein bodies of S. cepivorum differ from those ofother species in their metachromatic staining withtoluidine blue, suggesting that the protein theycontain is more strongly acidic.

Significant intracellular reserves of carbohydratecould not be detected. Glycogen is widespread as asclerotial reserve (Ergle, 1948; Aggab & Cooke,1981; Bullock et al., 1980; Backhouse & Willetts,1984) but is apparently not important in S.cepivorum. It is possible that intracellular carbo-hydrates are present as soluble sugars or polyolslike those found in Claoiceps and Myriosclerotinia(Cooke & Mitchell, 1969) but these cannot bedetected by the methods of the present study. Thecarbohydrate in medullary matrix is a potentialnutrient reserve although no evidence for itsutilization was seen.

Sclerotia from soil that had not had an onioncrop for 3 years showed considerable loss ofmedullary hyphae. Leggett & Rahe (1985) des-cribed disappearance of medullary tissue fromsclerotia incubated in saturated soil and suggestedthat this was related to enhanced rates of decay inwaterlogged soil. However most sclerotia in ourstudy showed no evidence of fungal or bacterialattack and their surviving hyphae appeared healthy.When bacteria were seen to be present, completedisorganization of hyphal contents had occurred.

The loss of reserves during prolonged burialsuggests that sclerotia do not remain entirelydormant but must undergo some physiologicalactivity. One aspect of this activity could be repairof the rind. The outside of the rind often containeddead or broken cells, similar to the collapsedsurface cells reported from scanning electronmicroscope studies of S. cepivorum by New, Coley-Smith & Georgy (1984). However, in all apparentlyhealthy sclerotia the rind still had at least one layerof live cells. This means that as cells die or becomedamaged they are replaced by differentiation ofnew rind cells underneath.

Observations on germinating sclerotia confirmedthe descriptions of Coley-Smith (1960) and New etal. (1984) and provided details of hyphal plugorigin. Hyphal growth occurred only from the cellsimmediately beneath the rind. There was noformation of primordia, such as those associatedwith carpogenic germination (Kosasih & Willetts,1975). This suggests that unless germination in thefield is significantly different from that in culture,anatomical changes are unlikely to occur duringthe period of conditioning required for host-induced germination. It also implies that thereceptive sites for germination stimulants are in thelayer of hyphal tips close to the sclerotial surface.

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(R eceived for publication 27 February 1987 )