NewPhytoi .S), 131, 3.

Apoplastic permeability of sclerotia ofSclerotium rolfsii^ Sclerotium cepivorum andRhizoctonia solani

BY NICOLA YOUNG AND ANNE E. ASHFORD

School of Biological Science, The University of New South Wales, NSW 2052, Australia

{Received 20 March 1995 ; accepted 29 May 1995)

SUMMARY

Intact mature sclerotia of Sclerotium rolfsii Sacc. and Sclerotium cepivorum Berk, produced in culture areimpermeable to the apoplastic tracer suiphorhodamine G. Both of these species produce sclerotia with rinds. Somemovement of suiphorhodamine into sclerotia of Rhizoctonia solani Kiihn, which have no rind, occurred butthe fluorochrome was arrested after permeation of at most the outer five layers of cells. In all cases, lowpermeability depended on an intact outer layer, and when sclerotia of each species were bisected to pro\ide directaccess of suiphorhodamine to all tissue layers, the Huorochroine permeated the cell walls and extracellular matrix(where present) of many cells within the scierotium. A marked reduction in permeability of intact sclerotia occursat maturity in a number of species and mi^ht be important in long-term survival.

Key words: Sclerotia, Sclerotium rolfsH, Sclerotium cepivorum, Rhizoctonia solani, apoplastic permeability.

INTRODUCTION

Scierotia are produced by many plant pathogens,and also by some mycorrhizal fungi (Dennis, 1980;Piche & Fortin, 1982; Grenville, Peterson & Piche,1985a, 6; Fox, \9Sba,b\ Moore, Ashford &Peterson, 1991). Those of pathogenic species havebeen more widely studied because they are importantpropagules of disease, resting in soil until a suitablehost is planted. Sclerotia usually survive for severalyears in the soil (Coley-Smith & Cooke, 1971) andhow they do so has been a focus of research to findnew methods of biological control. They are com-monly thought to be resistant to desiccation and thisresistance is usually attributed to the rind (Coley-Smith & Cooke, 1971 ; Willetts, 1971; Trevethick &Cooke, 1973; Bell & Wheeler, 1986; Cooke & Al-Hamdani, 1986; Willetts & Bullock, 1992). However,there are species that produce sclerotia withoutrinds, for example Rhizoctonia solani and VerticiUiumdahliae, and these have been shown to survive forsimilar long periods (Coley-Smith & Cooke, 1971).Sclerotia of the plant pathogen, Sclerotinia minor,become impermeable to the apoplastic tracer sulpho-rbodamine G as they mature and this coincides withthe deposition of pigment in the rind (Young &Ashford, 1992). This indicates that in these sclerotiathe development of the rind has a marked effect on

permeability to some molecules and thereforeperhaps contributes to the resistant properties of thesclerotium.

Using the same approach and techniques as for S.minor (Young & .'Xshford, 1992), sclerotia of threeother common plant pathogens have been tested fortheir permeability to suiphorhodamine G. Tbespecies were chosen to represent sclerotia of differentdegrees of complexity and from different fungal taxa.Sclerotium rolfsH has a sexual stage, Athelia rolfsii(Curzi) Tu & Kimbr. that belongs to the Basidio-mycotina (Tu & Kimbrough, 1978), lt bas highly-differentiated sclerotia w-itb an acellular 'skin' (amultilayered, pigmented rind), and a cortex differen-tiated from tbe medulla (Townsend & Willetts,1954). Sclerotium cepworum causes white rot ofAllium spp. (e.g. onion and garlic; Esler & Coley-Smith, 1983). This fungus has no known sexual statebut DNA, immunological and morphological analy-sis indicates that it is closely related to members ofthe Sclerotiniaceae (Novak & Kobn. 1991 ; Carbone& Kohn, 1993) in the Ascomycotina. The sclerotia ofS. cepivorum are very similar to those of S. minor butare much stnaller and have no cortex. The rind is oneto two cell layers deep and consists of rounded cellswith thick, darkly pigmented walls, whereas tbeinner medulla consists of closely packed cells withthinner, unpigmented walls (Backhouse & Stewart,

34 N. Young and A. E. Ashford

Figure 1. For legend see opposite.

permeability of sclerotia 35

1987). i?/ji£ctf^o«/a so/anMs an important pathogen ofcrops, with a wide host range (Letham, 1983 Q, b). Itis the asexual stage of Thanatephoris cucumeris(Frank) Donk, a basidiomycete (Tti, Kimbrough &Aldrich, 1977). Its sclerotia, however, are verydifferent from those of 5. rolfsii. At maturitj', theycomprise an undifferentiated mass of hyphae with nowell-defined zones. There is no rind, but the walls ofall hyphae within the sclerotium contain a brownpigment.

This work has provided the opportunity^ forcomparison of two basidiomycetes with one asco-mycete and a species closely related to an ascomycetefamily, as well as comparison between sclerotiawhich have a rind and those that do not. There is noprevious work that directly examines the perme-ability of sclerotia of any of these species although 5.rolfsii has been studied extensively (Chet, Henis &Mitchell, 1967; Chet & Henis, 1968; Chet, 1969;Nair et al, 1969; Trevethick & Cooke, 1973; Chet,Timar & Henis, 1977; Smith et at., 1989). Thegeneral features of the sclerotia are also described.

M.ATEBIALS AND METHODS

Fungal material

The isolate of Sclerotium rolfsii used was initiallycultured from soil in a wheat field near Narrabri,New South Wales, by C. A. Rugg of the RoyalBotanic Gardens, Sydney, The Sclerotium cepivorumisolate was cultured from soil near Hobart, Tasmaniaby Dr J. Wong and Rhizoctonia solani was initiallycultured from Papaya sp. by the Agriculture Schoolat the University of Sydney.

Cultures of S. rolfsii were maintained on potatodextrose agar at 27 °C in light, those of S. cepivorumon potato dextrose agar at 25 °C in the dark and R.solani on malt Marmite^ medium at 25 °C in thedark. Only mature sclerotia were used in exper-iments; these appeared in 5. rolfsii 17 d aftersubculture, in S. cepivorum after 14 d and in R. solaniafter 13 d.

Fluorochrome permeation experiments

A 0 2 mM solution of sulphorhodamine G (AldrichChemicals Lot no. 23, 056-1) in distilled water wasadded to Petri-dish cultures of each species so thathyphae and sclerotia were immersed in the fluoro-chrome for 1 or 3 h. A 1 h immersion time waschosen because in previous experiments this timehad proven sufficient to show sulphorhodaminepermeation patterns. An additional immersion timeof 3 h was used to determine whether sclerotia thatappeared to be impermeable to sulphorhodamineafter 1 h immersion still appeared so after 3 himmersion. In parallel experiments, sclerotia of eachspecies were removed from plates and bisectedbefore immersion in the sulphorhodamine solutionfor 1 and 3 h. Nine replicates were exatnined for eachspecies in each treatment except for S. cepivorum, forwhich there were five replicates in each treatment.

For controls, other Petri-dish cultures, or bisectedsclerotia from them, were immersed in distilledwater for 1 and 3 h then freeze-substituted as forsulphorhodamine-immersed sclerotia. Sclerotiawere also frozen directly from the Petri-dish cultureswithout any prior immersion in water or fluoro-chrome.

Freeze substitution

For structural work, sclerotia were sliced, frozenrapidly, substituted in dry acetone over a 3Amolecular sieve (Ajax) and embedded in LR whitesoft grade resin (London Resin Company) using themethod given in Young et al. (1993).

Sulphorhodamine-treated sclerotia and controlsfrom apoplastic tracing experiments were rapidlyfrozen, substituted at —70 °C in lOO'̂ o diethyl etherwithout fixative over a 3A molecular sieve andembedded in dry Spurr's resin (Spurr, 1969) underdry conditions as given in Young & Ashford (1992).To improve infiltration with the resin, specimens ofS. rolfsii and R. solani that bad been frozen intactwere bisected with a razor blade in the substitutionfluid after substitution and warming up, beforeinfiltration in the drv box.

Figure 1. Mature sclerotia of three plant pathogenic species viewed with a dissecting or light microscope.Median sections stained with toluidine blue O, pH 4 4 to show internal structure. Rind (r). cortex (c), medulla(m). extracellular matrix (e). {a) Sclerotium rolfsii scterotium with smooth brown surface (s). covered in someareas by a weft of unpigmented hyphae (w) (bar = 250//m). {b) Sclerotium cepivorum sclerotium; very small,spherical and black. In some places, the surface is covered by a weft of unpigmented hyphae (w) (bar =150//m). (r) Rhizoctonia solani sclerotium with irregular shape and variable brown surface. From the darkerbrown spots on the surface, a brown liquid is exuded (bar = 250 /im). (rf) Sclerotium rotfsii sclerotium with rindof brown-walled, tangentially flattened cells, well-developed cortex, and medulla that is looser at the centre ofthe sclerotium. The hyaline walls of cortical and medullary cells stain strongly with toluidine blue O. Thereis some extracellular matrix between cortical and medullary cells which contain reserves (arrows) (bar =20 Hm). (e) Sclerotium cepivorum sclerotium with relatively thick, black-walled rind and central medulla withextracellular matrix. Medullary cells contain reserve materials (arrows) (bar = 20//m). (J) Rhizoctonia solanisclerotium in section. The outer surface is on the right-hand side. All cell walls contain brown pigment but alsostain deep blue with toluidine blue. There is little differentiation of cells in the inner and outer regions and thereis no rind or cortex. Cells throughout the sclerotium contain reserves (arrows) (bar = 15//m).

36 A'. Young and A. E. Ashford

Figure 2. For lejijend set opposite.

Permeability of sclerotia 37

Sectioning and microscopy

Sections were cut at 2/^m on a Reichert-JungUltracut microtome. For all apoplastic tracing work,sections were flattened dry on to gelytin-coafedslides (Rogers. 1979), mounted in low-fiuorescenceimmersion oil and examined by fluorescence mi-croscopy with a Leitz Orthoplan Photomicruscopeunder green fxcjtation (Leitz filter combination BP530-560. HKP 580, LP 580). Photomicrographswere taken on Kodak Ektachrome 200 (da}-]ight)film.

For structural work, sections were flattened onwater droplets on to poly-L-lysine coated slides(O'Brien & McCully, 1981; van Noorden & Po)ak,1983), stained with 0-5 ",, toluidine blue O in sodiumacetate buffer, pH 4-4 for 15 min. mounted inimmersion oil, and viewed by bright field f)ptics witha Leitz Orthoplan Photomicroscope. Photomicro-graphs were taken on Kodak Technical Pan filmrated at 50 ASA.

The external appearance of sclerotia v. as examinedand photographed on Kodak Technical Pan filmrated at 50 ISO using a Wild M400 Photomakroscop.

H E S l ' t / l ' S

General features of mature sclerotia

Mature sclerotiii of Sclerotium rolfsii were 0-5-J-5mm in diameter, usually spherical and had asmooth brown surface covered in places by a weft ofunpifi^mented hyphae (Fig, 1 a). In sections, the outerrind was 2-3 cell layers thick and consisted oftangentially flattened cells with brt)\vn-pigmented

w alls (Fig. I h). l^hfs rind was surroundad b}' a 'skin 'of what is interpreted to be dried exudate and whichgave rhe sclerotia a smooth appearance. The cortexwas pseudoparenchymatous v '̂ith few interbyphalspaces, whereas the meduJla contained moreirregularly shaped hyphae with many interhyphalspaces. M(jst interh\-phal spaces were filled with anextracellular matrix that stamed strongly blue/greenwith toluidine blue O, indicating that it containedphenolics (O'Brien, Feder & McCully, 1964).

Mature scleroria oi Sclerofium cepivorum were330—420/vm in diameter, spherical and black (Fig.\ c). There was a rind 2-'3 cd] ]a>'ers thick of roundedcells with darkly pigmented walls, no cortex, and amedulla of slightly elongate cells with unpigmentedwalls and with many stained globular deposits insidethem fF'ig. 1 d). Most interhyphal spaces were filledwith an extracellular matrix. Cell walls and extra-cellular matrix stained a pale blue with toluidineblue, indicating the presence of phenolics.

Sclerotia of Rhizodoma wlam matured quickly.They varied greatly in shape and size, from roundedbodies 1-2 mm in diameter, to irregularly shapedbodies 3 ^ mm in diameter. They exuded a darkbrown liquid that coloured the growth medium andwere themselves an uneven brow'n, darker in areasfrom which the brown liquid had been exuded (Fig.\e). There was no obvious differentiation betw-eenh\'phae in different regions of the sclerotium andthere was no rind (Fig. ] /) . All hyphae were roundedand closely packed and had dark brown pigmentedwalls. There appeared to be some blue/greenstaining of the cell walls with toluidine blue but thestaining was masked by the wall pigment. Noextracellular matrix was detected in sections.

Figure 2. Sections o'l matuVL' intiicr or bisected sclcrotia of three plant pathogens frozen after immersion insulphcu-hodamine G or distilled water for 1 h or trozen directly from Petri-disb cultures. Rmd (r), cortex (c).medulla (m). [a) Sclerotium rolfsii sclerotium frozen directly. Walls (w). matrix and cytoplasm of all cellsautofiuorescf. Reserves autofluoresce inside cortical and medullary cells (bar = 10 ii.m). (b) Sclerotium rolfsiisclerotiuni immersed intatt in sulpliorhodamine Ci. Sulphorhodamme forms a thin orange layer (sr) outside thescarlet rind cells. No sulphorhodamint- is \i.sih)e iii.^ide the sclerotium (bar = 20//m), (r) Sclerotium cepivorumsck-rotium immersed intact in distilJed water. Medullary cell cytoplasm autofluoresces faintly. Noiiutofluori'scrncf was detected in cell wiills or extracellular matrix (bar = 15//m). id) Sclerotium cepivorumsclerotium immerst-d intact in sulphorhodamine CJ. Suiphorhodamine fluorescence is seen only in weft hyphae(wb) on the surface or rind cells (x) assumed to be dead. Inside the rind, onh' autofluorcscence of cortical andmedullary cell cytophism is seen (bar — 5 /'mj. (e) Rhizoctonia solam scleroriurn frozen directly. All cell walls(w) autofluoresce scarlet and some reserves autofluoresce faintly (bar=15/ /m) . (_/) Rhizoctonia solanisclerorium inimerst'd intact in Kulpborbodamine G. Sulphorhodamine fluorescence is seen only on the outersurface of some wails (arrow) and the inner surface of others (sr). Such cells are found only in the first four orfive cell hivers and inside these only autofluorescence of walls, extracellular matrix and reserves is seen. Theflocculfnt material near ihe surface (top of figure) is attached agar (a) that contains sulphorliodamine (bar =20/ml). (̂ 0 Scteriitiiiin inlfsii sclerotium bisected before immersion in sulphorhodamine G. Sulphorhodamineenters cell walls, cxrnitfHdlar mutr'is and protoplasts of medullary cells at the cut surface (p). Some walls tiearthe cut surface swell (t) and the cytoplasm correspondingly shrinks. Furthest from the cut surface, onlyautofluorescence iB seen (bar =20/mi) . (/') Bisected Sclerotium cepivorum sclerotium after immersion insulpborhodamme G. Sulphorhodamine fluoresces in cell walls (w) and extracellular matrix (e) and in somemedullary cell protoplasts (p) near tbe cut surface (bar = 5/mi), {i) Rhizoctonia solani sclerotium bisectedbefore immersion in suiphorhodamine d. Sulphorhodamine is seen in some walls (w), protoplasts of some cells(p) near tbe tiut surface and around the outer surface of some walls (sr). Beneath the first Hve cell layers fromthe cut .surface, un]> autofluorescencf is detected (bar = 20//m).

.̂ Young and A. E. Ashford

Permeation of sulphorhodamine G into intactsclerotia

In both the water-immersed and non-immersedcontrol sclerotia of Sclerotium rolfsii, bright scarlet/pink autofluorescence was seen in cell walls andextracellular matrix (Fig. 2a). Autofluorescence wasless bright in the medulla than in the rind and cortexand dullest in the extracellular matrix. Protein bodiesinside cells fluoresced red and the cytoplasmfluoresced a dull pink/red. In tbis species, sulpho-rhodamine fluorescence was distinguished fromautofluorescence by its colour rather than its in-tensity ; sulphorhodamine fluorescence was yellowishorange, whereas autofluorescence was scarlet or pink(Fig. 26). These colour differences were much easierto detect when viewed directly in the microscopethan in photomicrographs. Intact S. rolfsii sclerotiaimniersed in sulphorbodamine for 1 or 3 h beforefreeze substitution fluoresced brigbtly throughoutbut a comparison with controls showed tbat onlytbe orange fluorescence on the outer surface oftbe sclerotia was attributable to sulpborbodamine(compare Fig. 2a and 2b). No sulphorhodaminewas detected inside the rind of any mature,fluorochrome-immersed S. rolfsii sclerotia. Allsulphorhodamine fluorescence seen in these sclerotiawas confined to the weft hyphae and some apparentlydead rind cells.

Autofluorescence under green excitation w as muchlower in Sclerotium cepivorum and was seen only inthe cytoplasm of cortical and medullary cells (Fig.2c). Tbe cytoplasm of these cells fluoresced a paleorange that faded very quickly on exposure to greenexcitation. This made tbe autofluorescence difficultto record on film, but easy to distinguisb fromsulpborhodamine fluorescence, which did not fadeso rapidly. No autofluorescence was detected in cellwalls, including those of rind cells, or extracellularmatrix. AH mature sclerotia of S. cepivorum excludedsulpborbodamine during the 3 h treatment (Fig.Id). Tbe fluorocbrome was seen only in the weft ofhyphae tbat adbered to the outer surface of thesclerotium and in the lumen of some rind cells.These were interpreted to be dead. Faint auto-fluorescence was detected in cortical and medullarycells beneath the rind.

Cell walls of Rhizoctonia solani sclerotia auto-fluoresced a pinkish orange that became more pinkwitb prolonged exposure to green excitation but, onfllm, this appears bright scarlet (Fig. 2e). Insidesome cells, bodies resembling protein bodiesfluoresced a very dull red/pink. In a small number ofcells, the cytoplasm fluoresced a uniform dull pinkishorange (see Fig. 20- Sulphorhodamine fluorescencecould be distinguisbed from autofluorescence by itsorange rather tban red colour. Mature sclerotiaimmersed in sulpborbodamine for 1 or 3 h hadoccasional granular deposits of the fluorochrome

associated with cell walls of some peripheral byphaeand in the protoplast of occasional cells near these(Fig. 2/). Sulpborhodamine could also be detectedin cell walls of a few cells in the outer flve layers ofthe sclerotium. Elsewhere in the sclerotium. the onlyfluorescence was the autofluorescence of cell wallsand cell contents.

Permeation of sulphorhodamine G into bisectedsclerotia

In Sclerotium rolfsii sclerotia bisected before im-n\ersion in sulphorhodanaine, the fluorocbromepermeated most cell walls (Fig. 2^). Several med-ullary byphae along tbe cut surface and in tbe 4-8cell layers directly inside tbis, accumulated sulpho-rhodamine in the protoplast. This accumulatedsulphorhodamine was distributed evenly throughouttbe cytoplasm of tbe cells and fluoresced morebrightly in some organelles. In some cells near tbecut surface the cell walls appeared swollen and thecytoplasmic volume reduced.

When Sclerotium cepivorum sclerotia were bisectedbefore immersion in sulphorhodamine, fluoro-chrome permeated all cell walls and extracellularmatrix and fluoresced orange red. This fluorescencewas very dim away from the cut surface. Orangesulpborhodamine fluorescence was also seen inprotoplasts of most medullary cells at or near tbe cutsurface (Fig. 2h). The fluorochrome was distributedevenly tbrougbout the cytoplasm of these cells andalso accumulated m some organelles where itfluoresced more brightly.

In Rhizoctonia solani sclerotia bisected beforesulpborhodamme immersion, the fluorocbrome dis-tribution differed little from tbat in intact sclerotia.Sulphorhodamine was accumulated in many of tbeprotoplasts along the cut surface to a depth of threecell layers (Fig. 2i). Granular sulphorhodaminedeposits around cell walls of peripheral cells werealso frequent near the cut surface. Elsewhere in thescterotium below the 2-3 cell layers near the cutsurface, only autofluorescence of cell walls andcytoplasm was seen.

D I S C l • SS1O N

Of tbe three plant pathogenic fungi examined here,two produce sclerotia witb rinds. These sclerotia andthose of 5 . minor, wbicb also have a rind, areimpermeable to the apoplastic tracer sulpho-rhodamine G at maturity, despite considerabledifferences in their structure and morphogenesis,l^be earlier and more detailed study of Sclerotiniaminor showed tbat sclerotia of tbis species becomeimpermeable to sulphorbodamine as tbey matureand that reduction in permeability is correlated withdeposition of rind pigment (Young & Ashford,1992). The role of the rind as a primary barrier to

Permeability of sclerotia 39

movement of sulphorhodamine into mature sclerotiawas confirmed by experiments with damagedsclerotia, in which case the fluorochrome permeatedthe cortex and medulla, although movement was notas fast as would be expected for difFusion in water.This also happens in bisected sclerotia of the twospecies examined here, implying that the observedlack of permeation of intact sclerotia by sulpho-rhodamine is a property of the rind. It seems likelythat pigment deposition is the main factor conferringthis property on the rind. In all three species, thepigment involved is reported to be synthesized viathe same pathway from a 1,8-dihydroxynaphthaleneintermediate (Bell & Wheeler, 1986). The similarityof response of sclerotia of these three species toexternal application of sulphorhodamine implies thatlow permeability might be a common property offungal sclerotia. A comprehensive survey of sclerotiafrom a wide range of fungi would be valuahie inclarification of this point, as would examination ofvarious developmental stages ofthe scJerotia of eachfungus.

Sclerotia of R. solani differ from those of the otherspecies examined here in that they have no rind.These sclerotia are not completely impermeable tosulphorhodamine but, excepting the outermost fivecell layers, they exclude the fiuorochrome. Thisimplies that, although the rind provides an effectivebarrier to the movement of molecules such assulphorhodamine, it is not a rind that is necessary forreduced permeability but the presence of wallpigment. Cell walls of all cells in R. solani sclerotiacontain a pigment that appears to be formed via thesame pathway as those ofthe other species examinedhere (Bell & Wheeler, 1986). The cells throughoutthe sclerotium are closely packed and therefore ha\emost of the properties of a rind. Pigmentation of allsclerotial cell walls could be a disadvantage to thefungus; pigmentation of only the outer layers of thescierotium allows the inner hyphae to maintain theirplasticity. Such plasticity might be required in suchprocesses as rind regeneration after damage, in whichthe old medullary cells grown to form a new rind(Young, 1994), or during germination.

Sclerotia of all the species examined in this papersurvive for several years in soil and are resistant todesiccation (Townsend & Willetts, 1954; Coley-Smith & Cooke, 1971). Resistance to desiccation isoften attributed to the rind, but few explanations aregiven for how the rind does this. An increasedresistance to the movement of substances into thesclerotium could imply a resistance to loss of similarsubstances from the sclerotium. Although imper-meability to sulphorhodamine in no way impliesimpermeability to water, which is a much smallermolecule (only 18 Da compared with 553 Da forsulphorhodamine G), it is possible that water loss isalso reduced by pigmentation of the rind or out-ermost cell Javers,

Bisected sclerotia of all three species accumulatedsulphorhodamine in the protoplasts of hyphae nearthe exposed cut surface, as in S. minor (Young &Ashford, 1992). Such sulphorhodamine accumu-lation has been seen in cells of several plant systems(Canny, 1990) and has been attributed to enhancedproton transport such that the apoplast surroundingindividual cells or cell groups becomes relativelymore acidic than elsewhere. This neutralizesnegatively charged sulphorhodamine molecules,allowing them to cross the plasmalemma in thisregion. Sulphorhodamine accumulation in medul-lary cells of sclerotia appears to be a wound response.It is tempting to relate it to a rind regenerationprocess such as that shown in Sclerotinia sclerotiorum(Jones, 1 970) and S. minor (Young, 1 994), However,sulphorhodamine accumulation is also seen in R.solani w-here there is no rind to he regenerated andinner cells have heavily pigmented walls which mightnot retain plasticity. Whether the accumulation ofsulphorhodamine in these sclerotial protoplasts is aresult of reduction of extracellular pH by protonpumping is not clear at present.

.\ C K N O W L E D G E M E N T S

This work was financially supported by ARC funds andwas carried out while N'.Y. was in receipt of an AustralianPostgraduate Research Award. The authors thank BillAllaway for his comments on the manuscript.

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