Nezv Phytol. (1990), 114, 449-456

Ectomycorrhiza formation in Eucalyptus.

IV. Ectomycorrhizas in the sporocarps of the hypogeousfungi Mesophellia and Castorium in Eucalypt forests ofWestern Australia

BY B. D E L L \ N. MALAJCZUK^ T. S. GROVE'̂ AND G. THOMSON^

^School of Biological Sciences, Murdoch University, Murdoch, Western Australia 6150Division of Forestry and Forest Products, CSIRO, Private Bag, P.O. Wembley,

Western Australia 6014

{Received 12 October 1989; accepted 6 November 1989)

SUM MARY

Mesophellia and Castorium are common hypogeous macrofungi in the karri {Eucalyptus diversicolor F. Muell.) andjarrah {Eucalyptus marginata Donn ex Sm.) forests of south-western Australia.

Sporocarps of Mesophellia and Castorium develop 5-20 cm below the soil surface in close association witheucalypt roots. During differentiation of the sporocarps, eucalypt roots become trapped within the peridium wherethey branch profusely and form a dense ectomycorrhizal layer. Mature sporocarps of M. trabalis nom. ined.contain approximately 5 m of roots of 45 cm^ surface area. Anatomical studies have shown that these roots haveHartig nets penetrating to the hypodermis and are similar to the superficial eucalypt ectomycorrhizas formed in soiland litter. The association of Mesophellia and Castorium sporocarps with tree roots suggests that these areimportant mycorrhizal fungi in forests of southern Australia.

Key words: Mesophellia, Castorium, ectomycorrhizas, eucalypt forest, hypogeous fungi, sporocarps.

INTRODUCTION

Previous papers in this series have described thernorphology and anatomy of eucalypt ectomycor-rhizas synthesized in the laboratory or taken fromfeeder roots near the surface in forest soil (Malajczuk,Molina & Trappe, 1982; Malajczuk, Dell & Bougher,1987). Amongst the fungi which form symbioticassociations with roots of eucalypts are manyhypogeous fungi, those producing sporocarps belowground. Recently, Malajczuk, Trappe & Molina(1987) have drawn parallels between hypogeousfungi in eucalypt forests of Australia and related taxam coniferous forests of North America. However,many of the genera are unique to vegetation in eachof the respective continents.

Both Mesophellia and Castorium are predominatelyassociated with eucalypts in Australia. The sporo-carps are found at depth in the soil profile inter-twined amongst the tree roots. Although the sporo-carps of one of these genera are reported to containeucalypt roots (Beaton & Weste, 1983, 1984) little isknown of the mycorrhizal status of these roots. In

this paper we describe the structure of Mesophelliaand Castorium sporocarps and their internal ecto-mycorrhizas.

MATERIALS AND METHODS

Sporocarps for histological examination were col-lected from karri {Eucalyptus diversicolor F. Muell.)and jarrah {Eucalyptus marginata Donn ex Sm.)forests in south west Australia. After dissection smallpieces were fixed in 3 % glutaraldehyde in 25 mMpotassium phosphate bufier (pH 7-1) or Karnovsky'sfixative. Sections were prepared from epoxy em-bedded material. Except for the Masson Fontanamethod (Bancroft & Stevens, 1977) techniqueswere the same as described previously (Malajczuket ah, 1987).

RESULTSEield observations

The most common species found during the earlysummer sampling period (December) were Meso-

450 B. Dell and others

Figure 1. Structure of the sporocarp of Mesophellia trabalis. (a) Immature sporocarp with central glebal core(C) prior to differentiation of trabeculae within the outer gleba (G). The root layer (arrows) is present in theperidium (P). (h) Mature sporocarp dissected to expose the multilayered peridium (P) surrounding the sporemass (S). P|, ectoperidium, P ,̂ mesoperidium, P ,̂ endoperidium. (c) Detail of mature peridium with prominentliving root layer (R). The spore mass has been removed exposing the trabeculae (T). The heavily pigmentedmain ectoperidial layer (P;,,) is surrounded by a distinct layer of cottony hyphae and soil particles {P^J. (d)Mature sporocarp cut open exposing the peridium (P), spore mass (S) and columella (C) in a live specimen,(e) Section through the immature outer gleba with differentiating hymenium (HY). (/) Section through themature hymenium with basidia bearing spores (arrows). (e,f) Stained with SchifT's Reagent/toluidine blue.

Ectomycorrhiza formatioti in Eucalyptus. IV 451

Figure 2. For legend see p. 453.

452 B. Dell and others

(f) 5 (xm

Figure 3. For legend see facing page.

Ectomycorrhiza formation in Eucalyptus. IV 453

phellia trabalis nom. ined., M. labyrinthina nom.ined. and Castorium camphoratum nom. ined.Sporocarps were readily recognised from gravelpisoliths because of their characteristic round shapeand low density. Sporocarps were abundant infegrowth and mature forests and were located belowthe litter layer in the mineral soil to a depth of 20 cm.As many as 34 sporocarps/m^ were observed at somesites.

Sporocarp structure

Peridium. The sporocarps of Mesophellia andCastorium form from localized aggregations ofTiycelia within the soil horizon. As the peridium andgleba begin to differentiate, eucalypt roots grow andbecome embedded within the peridium (Fig. 1 a).The sporocarps are thus connected to host trees byroots. Hypbae from the sporocarps ramify throughthe soil and form ectomycorrhizal associations witheucalypt roots in the mineral soil. The maturesporocarps are characterized by a firm, crustyperidium surrounding a central core {Mesophellia)and spore bearing cavities {Mesophellia andCastorium) [Figs 1 d; 2{a, c)]. The fully differentiatedperidium is three-layered in Mesophellia andCastorium (Beaton & Weste, 1983, 1984). In thispaper we designate the exoperidium as Pj, meso-peridium as P,̂ and endoperidium as P3. In M.trabalis the P, and P3 each consist of two layers [P^^,

It)' P3a> P31,; Fig. 1 (a, b)] and in M. labyrinthina (Fig.2o) PJ is two-layered. In C. camphoratum [Fig. 2(b,«)] the P3 is two-layered. Eucalypt roots are confinedto a single wbite, rubbery layer within Pj [Figs. 1 c;2(6,e)]. The bulk of the peridial tissue consists,however, of closely packed hyphae (Fig. 2h). Oc-casional small woody roots (1-1-5 mm diam) passirom the soil through the outer dark brown or black-Pigmented peridial layers (Pj^ in Mesophellia, Pj inCastorium) and connect with the fine root clusterswithin the peridium. Many of the fully mature

sporocarps sampled were lying unattached in thesoil. It is not clear at what stage of development rootconnections with the host become detached. Liveroots have, however, been observed in sporocarpswith spores present.

In the two Mesophellia spp. the outermost layers(Pj) of the peridium consist of a loose hyphaltomentum containing soil debris and scatteredeucalypt ectomycorrhizas. In Castorium the outer-most peridial layer (Pj) turns black and hardens asthe fruiting body matures [Fig. 2{e,g)]. Both the Pjlayer and the inner endoperidium (Fig. 2/, P3t,)reacted with ammoniacal silver nitrate in the absenceof an external reducing agent suggesting melaniz-ation had occurred.

Gleba. In M. trabalis the outermost part of the glebadifferentiates into wedge shaped trabeculae whichconnect the rubbery core to the peridium [Fig. 1 (c,d)]. The basidia develop between the trabeculaewithin elongated channels [Fig. \{e,b)]. In M.labyrinthina the basidiospores form within labyrin-thine channels which transect the gleba (Fig. 2b).Spores in Castorium camphoratum occur in numerouslocules through the gleba (Fig. 2c).

Sporocarp ectomycorrhizas

Eucalypt roots which develop within the peridium ofMesophellia and Castorium [Fig. 3 {a-c)] are closelypacked and in their dimensions resemble fine rootsfound in soil or litter. In M. trabalis each sporocarphas on average 5 m of short roots, giving a surfacearea of approximately 45 cm^. All of the fine roots areectomycorrhizal since they have a well developedHartig net penetrating between unextended epi-dermal cells to tbe hypodermis [Fig. 3{d,e)]. Ex-ternally, the epidermis is completely surroundedby closely packed fungal hyphae thus making itimpossible to distinguish a mantle from other peridialtissue.

Figure 2. Structure of the sporocarps of Mesophellia labyrinthina (a,b) and Castorium camphoratum (c~h). (a)Mature sporocarp cut open exposing the peridium (P) and gleba (G). (b) Mature sporocarp showing the 2-layered exoperidium (Pj) and the cartilaginous core (C) of the gleba penetrated by labyrinthine locules (arrows).The mesoperidium (P^) which lies internal to the dark-brown pigmented layer (Pj) is densely packed with roots.{c,d) Mature sporocarp cut open exposing the peridium (P) and inner gleba (G). The melanized exoperidiuni(PJ) surrounds the root-filled mesoperidium (Pj)- The endoperidium (P3) is defined internally by a narrowpigmented zone (-fl^). (e,f) Sections through the mature peridium: P^, outer crusty, thick melanised layer; Pj,root occupied zone, Pj^, rubbery pseudo-parenchymatous layer; P,,,, inner melanised layer of the endoperidium;R, roots, {g) Junction of ecto- and meso-peridial layers. Scattered hyphae (arrows) can be seen in the crustylayer, {h) Hyphal morphology of endoperidium (Pj^). (e, g, h) Stained with toluidine blue, (/) Masson-Fontana.

Figure 3. Structure oi Eucalyptus roots from within the peridium of Mesophellia trabalis (a,f), M. labyrhithina(b) and Castorium camphoratum {c-e). (a) Surface view of root clusters (arrows) after removal of the outerperidial layers, {b, c) Transverse sections of root clusters showing the unbranched superficial ectomycorrhizas(R) embedded in fungal tissue (-̂ AT)- (d) Detail of ectomycorrhizal anatomy. The Hartig net (arrows) extendsbetween the unexpanded epidermal cells (E) as far as the outer cortex (H). {e) Section of senescingectomycorrhizas showing invasion of cortical cells (CX) by fungal tissue (arrows). (/) Portion of ectomycorrhizashowing the Hartig net (HN) penetrating to the hypodermis (H). Many fungal hyphae (-jlf) lie external to theepidermis (E) in the region where the mantle occurs in soil-dwelling superficial ectomycorrhizas. Electron-dense material is present in interhyphal areas (arrows), {b-e) Stained with toluidine blue.

454 B. Dell and others

(d)Figure 4. Structure of ectomycorrhizas. (a) Portion of the Hartig net (HN) and mantle (M) in a matureectomycorrhiza taken from the peridium of Castorium camphoratum, showing the barrier zone within thehypodermis due to the deposition of electron-dense material (*) and suberization of the hypodermal cell wall(arrows). (6) Hypodermal cell wall with suberized lamellae (small arrows) adjacent to a vacuolated hyphalcomponent of the Hartig net (HN) and two epidermal cells (E). The middle lamella between the hypodermaland epidermal cells has separated (large arrows), (c—e) Mature Eucalyptus diversicolor ectomycorrhizas takenfrom forest soil adjacent to sporocarps of Mesophellia trabalis. (c) Transverse section showing the narrowsheating mantle (M) and well developed Hartig net (arrows) extending between vacuolated epidermal cells asfar as the partially collapsed hypodermis (H). The cortex (CX) has lignified cell walls, (rf) Electron-densematerial (*) in the inner mantle (M) adjacent to the epidermis (E). (e) Partially vacuolated hyphae in the Hartignet (HN) between two epidermal cells (E). (c) Stained with toluidine blue.

Ectomycorrhiza formation in Eucalyptus. IV 455

As in similar superficial ectomycorrhizas{Malajczuk et al., 1987), the hypodermis developschemical (polyphenol accumulation) and physical(suberized wall lamellae) barriers to further pen-etration by fungal tissue [Fig. 4 (a, 6)]. However, innnature sporocarps, the cortex may become invadedintracellularly, presumably during senescence of theroots.

Comparison of sporocarp and soil ectomycorrhizas

Eucalypt fine roots synthesized in the laboratory andfrom beneath M. trabalis in the karri forest wereexamined for comparison with ectomycorrhizasobserved in the sporocarps. The soil-borne roots aresimple or weakly pyramidally branched and areenclosed in a loosely packed, thin mantle [Fig. 4(c,d)]. Anatomical features of these superficial ecto-mycorrhizas [Fig. A{d,e)'\ are similar to those in theectomycorrhizas observed within the peridial layers(Fig. 3).

DISCUSSION

Mesophellia and Castorium sporocarps were alwaysassociated with first and second order lateral roots ofeucalypts. These roots were enveloped in sheaths ofmycelium which extended into the soil, thus formingaggregations of soil, mycelia, fruit bodies and treeroots. During diflFerentiation of the sporocarps ofMesophellia and Castorium eucalyptus roots becometrapped within the peridial layers where they branchprofusely and form a dense ectomycorrhizal layer. Incontrast to Beaton & Weste (1983), most of the shortroots enclosed in the mesoperidia had an Hartig neteven where external deposits of phenolic materialsWere present. Since the sporocarps probably takeseveral months to mature before all root connectionswith the host are severed, these ectomycorrhizas mayplay a role in nutrient relationships between the hostand tree. The fungus could for example stimulateroot development within the peridium thus en-hancing additional carbohydrate flow into the sporo-carp. In addition, uptake of inorganic nutrients bythe tree may be enhanced by placement of rootswithin the fungal tissue. In other genera such asHysterangium which also cohabit the eucalypt forest,tree roots are excluded from the sporocarps, pre-sumably due to chemical as well as physical barriers.In Mesophellia and Castorium the roots fail topenetrate the gleba and are restricted to a narrowzone within the peridium [e.g. Fig. 2(e,/)]. The lackof penetration of adjacent layers of the peridium (e.g.P31 Fig. 2d) is puzzling given the absence of anyobvious barrier zone. Sporocarps at the stage ofgleba and peridium differentiation were not availablein this study so it remains unclear how the timing ofsporocarp development relates to root growth.

Beaton & Weste (1983) did not observe roots inimmature sporocarps and concluded that rootsinvade near maturity. More likely, peridial de-velopment and root extension are synchronizedevents.

Mesophellia and Castorimn are unusual in thatsporocarps are abundant in early summer when mostof the other fleshy hypogeous and epigeous groupshave finished fruiting (Christensen, 1980). Twofactors appear to be important: firstly, the sporocarpspossess a crusty outer peridium which probablyresists dessication, and secondly, they are found atdepth (some 5-20 cm below the soil surface).

Malajczuk et al., (1987) have identified inter-actions between eucalypts, hypogeous mycorrhiza-forming fungi such as Mesophellia, and smallmycophagous mammals. Much of the sporocarptissue is consumed by small marsupials. Thehypogeous fungi are particularly dependent onanimals for their dispersal (Trappe & Maser, 1977).Spores oi Mesophellia that pass through the digestivetracts of mycophagous marsupials remain viable andhave been shown to form ectomycorrhizas witheucalypt seedlings in glasshouse trials (Lamont,Ralph & Christensen, 1985).

The frequent occurrence of Mesophellia andCastorium sporocarps in eucalypt forests in southernAustralia (Malajczuk, unpublished observations)suggest that these are important mycorrhizal fungi innatural forest stands. Detailed samplings are re-quired to obtain information on the magnitude ofinorganic and organic nutrients turned over by thesporocarps.

ACKNOWLEDGEMENTS

We thank Joanne Robinson for the line drawings and forhelpful comments.

REFERENCES

BANCROFT, J. D. & STEVENS, A. (1977). Theory and Practice ofHistologicat Techniques. Churchill Livingstone, Edinburgh.

BEATON, G . & WESTE, G . (1983). The genus Mesophellia inVictoria, Australia. Transactions of the British MycologicatSociety 80, 209-211.

BEATON, G . & WESTE, G . (1984). Victorian hypogaean gastero-mycetes: Mesophelliaceae. Transactions of the British My-cological Society 82, 665-671.

CHRISTENSEN, P. E. S. (1980). The biology of Bettongia penicillata(Gray, 1937) and Macropus erigenii {Desmarest, 1817) in relationto fire. Forest Department Western Australia Bulletin 91, 1-90.

LAMONT, B . B., RALPH, C . S . & CHRISTENSEN, P. E. R. (1985).Mycophagous marsupials as dispersal agents for ectomycor-rhizal fungi on Eucalyptus calophylla and Gastrolohium hilolntm.New Phytologist 101, 651-656.

MALAJCZUK, N . , MOLINA, R . & TRAPPE, J. M. (1982). Ectomycor-rhizal formation in Eucalyptus. \. Pure culture, synthesis, hostspecificity and mycorrhizal compatibility with Pinus radiata.New Phytologist 91, 467-482.

MALAJCZUK, N . , DELL, B . & BOUCHER, N . L . (1987). Ectomycor-rhiza formation in Eucalyptus. IH. Superficial ectomycorrhizasinitiated by Hysterangium and Cortinarius species. New Phy-tologist 105, 421-428.

456 B. Dell and others

MALAJCZUK, N . , TRAPPE, J. M . & MOLINA, R. (1987). Inter- TRAPPE, J. M . & MASER, C. (1977). Ectomycorrhizal fungi:relationships among some ectomycorrhizal trees, hypogeous interactions of mushrooms and truffles with beasts and trees,fungi, and small mammals: Western Australian and north In: Mushrooms and Man. an inter-disciplinary approach toviestern Amenc&n para\\e\&. Australian Journal cf Ecology \2, mycology (Ed. by T.Walters), pp. 165-179. Linn, Benton53-55. Community College, Albany, Oregon.