New Phytol. (1995), 129, 4.19-447

Ericoid mycorrhizas and rhizoid-ascomycete associations in liverworts sharethe same mycobiont: isolation of thepartners and resynthesis of the associationsin vitro

BY JEFFREY G. DUCKETT^ AND DAVID J. READ-

^ School of Biological Sciences, Queen Mary and Westfield College, Mile End Road,London, El 4NS, UK"^Department of Animal and Plant Sciences, University of Sheffield, Sheffield, SIO 2TN,UK

{Received 8 July 1994; accepted 4 October 1994)

SUMMARY

The hypothesis was tested that rhizoids of leafy liverworts of the families Lepidoziaeeae, Calypogeiaeeae,Cephaloziaceae and Cephaloziellaeeae can he infected hy the aseomycetous fungal endophyte Hvmeiioscyplnisericae which forms erieoid mycorrhiza with the major ericaceous genera Caliiina, Erica, Rluxtodeiidron andVaecinium. The extent of the specificity of any such association was also examined by growing the liverworts withpure cultures of the putative aseomycetous ericoid endophyte Oidiodendron, with the basidiomycetous endophyteof orchids, Ceratobasidiiim cornigeriiiii, and with several ectomyeorrhizal fungi.

It was confirmed that the memhers of these li\-erwort families tested, most of which are associated in nature withericaceous plants, were readily infected by H. ericae, as well as hy isolates obtained from the liverworts themseh-es.The latter, when used to challenge aseptically grown seedlings of the erieaceous genera on water agar, producedtypical ericoid mycorrhiza. Neither the Oidiodendron isolates nor the orehid or eetomyeorrhizal fungi infected theliverworts, and all failed to reproduce the characteristic swelling of the rhizoid tips which is seen in nature andin plants inoculated with H. ericae.

The results provide a major extension of the likely host range o( H. ericae and may liave eonsiderahle significancefor hoth groups of plants which, as a consequence of sharing the same endophyte, have the potential to act assourees of inoculum for each other in nature. The possible physiologieal and ecological impacts of the associationfor hoth liverwort and erieaeeous hosts are discussed.

Key words: Leafy liverwort, rhizoid infection, ericoid mycorrhiza, ascomycete, shared hosts.

INTKODUCTION to, tliose reported to form ericoid mycorrhiza in thehair roots of major ericaceous genera such as Calluna,

I'he widespread occurrence of aseomycetous fungi Erica, Rhododendron and Vaecinium. In addition toin the rhizoids of leat'y liverworts of the families the structural and ultrastructural similarities be-Lepidoziaceae, Calypogeiaeeae, Adelanthaceae, tween the hyphal coils formed in the swollen rhizoidCephaloziaceae and Cephaloziellaeeae has been con- apices and flagellifortn axes of the leafy hepatics atidfirmed by ultrastructural (Duckett, Renzaglia & Pell, those formed in epidermal cells of the hair roots of1991) and cytological (Duckett & Read, 1991) the ericaceous hosts, the habitats in which the twoexatnination of species representative of both tem- associations occur show remarkable overlap,perature and tropical ecosystems. Several features of Whether in the moist acidic peats and mor-humusthese associations suggest the possibility that the soils of the boreal atid temperate zones, or infungi involved may be the same as, or closely related epiphytic cloud forest cotnmunities of tropical

440 y. G. Duckett and D. J. Read

mountains (Pocock et ah, 1984) ericaceous plants ofthe genera listed above tiormally, if tiot invariably,grow together with liverworts with this type ofinfection. The ecological implications of a sharedfungal association would be considerable, especiallyperhaps for the ericaceous platit, the seeds of whichin tiature would normally germitiate in close prox-imity to hepatics bearing appropriate inoculum.

Since the aseomycetous ericoid mycorrhizalfungus Hymenoscyphus ericae and the endophytes ofliverwort rhizoids cati readily be grown axenicalJy, itis evidently desirable to carry out reciprocalresynthesis experiments to examine the possibilitythat the two unrelated groups of autotrophs share acommon fungal associate. Clearly also, the extent ofspecificity of any such relationship should also beexamitied by growing liverworts with other putativeendophytes.

Resynthesis, which, as pointed out by Leake &Read (1991), is one of the major prerequisites for thesatisfaction of Koch's postulates, has previouslyenabled confirmation of the ability of H. ericae toform ericoid associations in Galluna (Pearson &Read, 1973 ; Bonfante-Easolo & Gianinazzi-Pearson,1982), Erica (Duclos, Pepin & Bruchet 1983; Read,1983), Rhododendron (Duddridge & Read, 1982) andVaecinium (Stribley & Read, 1974, 1976, 1980).Associations which resemble those produced byH. ericae can also be obtained by inoculatioti withOidiodendron spp. (Dalpe, 1986, 1991; Douglas,Heslin & Read, 1989; Currah, Tsuneda & Murakami1993).

Considerable specificity in the relationship be-tween H. ericae and its host plants is suggested bythe observation that antagonistic effects are readilyproduced when 'non-hosts' are inoculated with thefungus, irrespective of whether they are distantly(Gianinazzi-Pearson, 1985) or closely (Giovanetti &Lioi, 1990) related to its true hosts.

In the present study we test the hypothesis thatcommonly co-existitig ericaceous and leafy liverwortspecies may share the same fungal endophyte.Reciprocal resynthesis experiments are carried outusitig known and putative ericoid mycorrhizal fungi,other ascomycetes that do not form ericoid mycor-rhiza and some ectomycorrhizal basidiomycetes.

MATI-IUALS AND METHODS

Axenic cultures of a variety of liverworts (Tables 1and 2) were established from spores, gametophyteapices or gemmae on Parker agar (Klekowski, 1969)following surface sterilization for 30 s- 2 miti inL5% sodium hypochlorite solution containing adrop of detergent. Cultures were maintaitied at roomtemperature (18-22 °C) or at a temperature of 10 °Cunder illuminatioti from fluorescent lights givitig atiirradiance of approximately 5 W m" s"^

Stems of Calypogeia jissa and subterranean axesof Kurzia paueijiora, Cephalozia bieuspidata, C.leuca?ttha, Odontoschisma deniidattitn and O. sphagnibearitig tiumerous fungus-containing rhizoids wereseparated trotn their native substratum by thoroughwashing in distilled water and any adherent organicmatter was i-emoved with a fine paint brush.Eollowing 5 s immersion in hypochlorite andseveral rinses in sterile distilled water the axes wereplanted on 2 "o malt or tiutrient-free agar at roomtemperature. Hyphae grew out from the rhizoidswithiti a ten day period on both media. They weresubcultured, and subsequently used to inoculate theaxenic liverwort cultures or axenic seedlitigs of Ericaeinerea, Calluna vidgaris and Vaecinium corymbosttmL. which had been established from surface-sterilized seeds.

Axenic ericaceous seedlings and samples of theaxenically cultured liverworts were also added toagar cultures of the following known mycorrhizalfungi: Hymenoscyphus ericae (Read) Korf. & Kernen(isolates 100 atid 101 from Calluna vulgaris) Oidio-dendroft griseum Robak (from Vaecinium corym-bosum), O. maius Baron and Cenococcum geophilumEr. [both from Picea sitchensis (Bong.) Carr], Suillusboviniis (Er.) O. Kuntze and Anianita musearia (L. exEr.) Llooker (both from Pinus syhestris L.) Paxillttsinvolittus (Er.) Er. (from Betula pubescens Ehrh.) andCeratobasidium eornigertun Rogers [from Goodyerarepens (L.) R. Br.]. Eull details of these isolates aregiven in Duckett & Read (1991).

Cleaned specimens of the following liverworts:Cephalozia hiberniea, C. lunuUfolia, Cladopodiellajhiitans, Kurzia sylvatiea, K. trichoclados, Odoti-toschisma prostratum, Mylia atiomala and Telaraneanematodes, which typically contaiti rhizoidal asco-mycetes (Table 1) were placed in Petri dishescontaining either distilled water, Parker nutrientsolution or damp silica sand and the culturesmaintained over periods of several months under thelaboratory conditions detailed above. All new growthwas fungus-free (Duckett et al., 1991). At regularinter\'als these cultured plants plus those of theaxenically-grown liverworts were returned to theirorigitial substrata or foreign natural substrata ('liable1) maintaitied in the laboratory in plastic boxeslightly covered with polythene, and their furtherdevelopment monitored.

Living specimens were mounted in water andobserved and photographed under differential in-terferetice contrast optics with a I^eitz Ortholuxmicroscope. Some of the materials were also stainedin 5 //g mL' 3,3'-dihexyloxacarbocyanine iodide(DiOC5(3)) for 5 min and observed by epi-fluorescetice optics using a blue excitation filter(Duckett & Read, 1991).

Nometiclature lor the British hepatics followsSmith (1990) and the hepatic classification is that ofSchuster (1984).

Ericoid mveorrliizas and liverzvort rhizoid-ascomvcete associations 441

Table 1. Sourees of liverworts forming rhizoid-ascomycete associations

Original locality and substrate Hepatics present Ericaceae present

(1) Valley bog peat, Cranesmoor, NewForest, England

(2) Raised hog peat, Woodfield, CentralIreland

(3) Sandstone rocks, E. Susse.x. lingland

(4) Qiierciis woodland, Co. Kerry, Westot Ireland

(5) Rotten logs, Merioneth, N. Wales

(6) Peaty soil beneath Rhododendron,S.E. l-:ngland

(7) Peaty soil, heathland f'ast Anglia,England

(8) Peaty soil, ravine Merioneth N.Wales

(9) Rock faee. Red l<"'ork I-\ills, E.Tennessee, USA

Kurzia paueijioraCalypogeia jissaCephalozia bieuspidataC. cimnivensOdontoseliisma sphagniCladopodiella jhiitansKurzia paueijioraK. sylvatieaCalypogeia jissaCephalozia leucaiitliaC, bieuspidataC, loitlesbergeriOdontoschisma sphagniO. denudatiimMylia aiiomalaLepidozia reptansKurzia sylvatieaCalypogeia muelleraiiaCephalozia bieuspidataOdontoschisma deiiiidatumCeplialoziella diziaricataKurzia trichocladosTelaranea nematodesCalypogeia miiellcranaCephalozia hiberniea

Lepidozia reptansCalypogeia fissaCeplialozia bieuspidataC. liiindijoliaNowellia curvifoliaOdontoschisma deiiiidatiimLepidozia reptansCalypogeia fissaCephalozia biciispidalaCalypogeia jissaCephalozia bieuspidataCeplialoziella spp.Cladopodiella jraiHisciLepidozia reptansCalypogeia jissaC. muelleraiiaCeplialozia bieuspidataOdontoschisma prostratum{SW.) Trev.Kurzia sylvatieaCalypogeia nmelleratia

Calluna vulgaris (L.) HullErica tetralix L.Vaecinium oxvcoeeos(Rupr.) Hook.

Calluna vulgarisErica tetralixVaecinium oxyeoccos

Calluna vulgarisErica einerea L.Vaecinium niyrtillus L.Rhododendron poiiticum L

Calluna vulgarisErica einereaVaeeiniiim myrtillusV. vitis-idea L.Rhododendron poiitieiimArbutus uiiedo L.

Rhododendron poiitieum

Calluna vulgarisErica einerea

Calluna vidgarisVaecinium myrtillus

Kalniia latijolia L.Gaidtlieria prociimbeiis L.Leueotboe axillaris D. Don.Rhododendron maximu7n L.

O H S E R V A T t O N S

As with ericaceous hair roots (Pearson & Read,1973), when surface-sterilized liverwort stems bear-ing asconiycete-intected rhizoids are planted onnutrient-free agar, within 5-10 d numerous h>-phaegrow out into the medium (Eigs 1, 2). After 2-3 wkthe hyphae extend throughout the mediutii iti 5 cmPetri plates atid when subcultured grow with thesame vigour.

When axenic seedlings of the Ericacece are plantedonto this futigus-containing medium the hair rootshecome infected in e.xactlv the same manner as those

grown in the presence of Hymenoscyphus ericae inculture (Read, 1983) and those found in nature.Scattered h>phae are present in the mucigelassociated with the root caps in both wild hair roots(Eig. 3) and those grown in the presence of the fungifrom lixerworts (Eig. 4). Behind the apex in bothwild (Eig. 5) and experimentally infected hair roots(Eigs 6-8) the fungi grow over the surface and formextensive coils within the epidermal cells. Both thesurface hyphae and those within the epidermal cellsstain intensely with DiOC,j(3) (Eigs 9, 10).

1 'igures 11-26 are representative illustrations ofli\'erwort rhizoids grown in the laboratorv in the

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Ericoid mvcorrhizas and liverivort rhizoid-ascomvcete associations 443

Figures 1-8.

Figures 1 and 2. Kurzia pauciflora. Hyphae growing into water agar from the swollen tips of infected rhizoids,x410. Figure 3. Erica cinerea, tip of a wild hair root showing fungal hyphae in the mucigel; cells of therudimentary root cap are arrowed, x 200. Figure 4. Calluna vulgaris, tip of hair root grow-n on water agar inthe pi'esence of the fungus from Kurzia pauciflora showing hyphae in the apical mucigel. The root cap isarrowed, x 220. Figure 5. Calluna vulgaris, hyphae in the epidermal cells of a wild hair root, x 610. Figures6 and 7. Details of hair roots of Calluna vulgaris infected with the fungus from Kurzia. Figure 6. Internal andexternal hyphae, x 470. Figure 7. Extensive development of hyphae in two adjacent epidermal cells, x 600.Figure 8. Hair root of Vaccinium corymbosum showing extensive infection of the epidermal cells with the fungusfrom Kurzia, x 220.

444 J. G. Duckett and D. J. Read

Figures 9—17. For legend sec opposite.

Ericoid mycorrhizas and liverivort rhizoid-ascomycete associations 445

presetice of a variety of fungal isolates. Pictures ofnaturally-occurritig futigus-infected rhizoids of thesame species may be found in Duckett et al. (1991)and Duckett & Read (1991). Whereas in the presenceof Flymenoscyphus the majority (over 90%) of therhizoid tips become swollen and packed with hyphae(Figs 11, 13-17) when grown together with all theother futigi the rhizoids remain cylindrical (Figs 12,18, 19).

When compatible liverworts are grown fromspores oti /f3^?Kewo.';c3'/)/;M -containing medium (Figs20, 21) the flrst cells to become infected are mucilagepapillae distributed along the filamentous sporelitigs.Stages itl the infection process in mature rhizoids areshown in Figures 22-25. Initially the hyphae growaround the surface of the rhizoids (Fig. 23) mostlikely within the superficial mucilage pockets (Fig.22). Subsequetitly penetration is marked by acessatioti iti rhizoid elongatioti atid increasing dis-tortion of the tip, though in NowelUa this is morepronounced in wild-collected specimetis (Fig. 26)than in culture (Fig. 24). The majority of the fungalpenetration sites are near the rhizoid apices (Duckettet al,, 1991). Here the hyphae are tightly coiledwhereas their growth is straight iti the shafts (Fig.25). As itl nature, in the resynthesized associationsthe futigi do tiot extend beyond the bases of therhizoids itito the medullary cells of the hepatic stems.

The results of all the resynthesis experiments aresummarized in Table 2. The fungal isolates from thesix difl'eretit hepatics re-infected tiot only the origitialspecies but also all the other species known to formrhizoid-ascomycete associations in tiature. Iti ad-dition, all six hepatic isolates formed typical hair-root mycorrhizas with all three species of theEricaceae. Iti strikitig cotitrast neither Hymeno-seyphus nor the hepatic isolates petietrated livingrhizoids of atiy of the liverworts which in nature areeither futigus-free or contain basidiomycetous orzygomycetous etidophytes. All the other tnycorrhizalfungi failed to fortn rhizoid associatiotis with all theliverworts tested.

When the a.xetiically-cultured or fungus-freehepatics were returned to their native substrate or toforeign tiatural substrata (Table 1) their new rhizoidsdeveloped asconiycete associatiotis itidistinguishablefrom those found originally or resynthesized in

culture. Particularly notable among these results wasthe formatioti of rhizoid-futigus associations in: (1)Odontoschisma prostratum, Calvpogeia nutelleranaatid Kttrzia sylvatiea of North Americati origiti otiBritish substrata; (2) the bog species Mylia anomalaand Cladopodiella fluitans oti rotteti logs and soilfrom beneath Rhododendrons far removed from theirnatural habitat and (3) Noivellia curvifolia, tiormallya rotteti log species, on terrestrial peaty soil.

DISCUSSION

The results provide confirmatioti of the hypothesisthat the rhizoids of a range of leafy liverwort speciesof the fatiiilies Lepidoziaceae, Calypogeiaeeae,Cephaloziaceae and Cephaloziellaeeae cati be iti-fected by the ericoid mycorrhizal fungus Hymeno-scyphus ericae, which produces in their swolletiapices hyphal coils typical of those seen in nature.They also provide a very considerable increase in thelikely host range of this fungus. An apparentlysitnilar ascotnycete futigus has been isolated fromrhizoids of Ceplialoziella exiiflora growing in An-tarctica (Williams, Roser & Seppelt, 1994).

As far as we are aware, the present study is the firstreport of the same fungus infectitig both vascularatid non-vascular plants. This discovery clearlyinvites in\'estigation into the possibility that sotne ofthe zygomycetous and basidiomycetous fungiassociated with other hepatics (Boullard, 1988;Ligrotie & Lopes, 1989 ; Ligrotie, Pocock & Duckett,1993) may be, respectively, VA and ectomycorrhizal(or orchid) mycorrhizal fungi of higher plants. Atflrst sight one highly promising pairing is that of theparasitic achlorophyllous hepatic Cryptothalliisinirabilis growing in close proximity to the roots ofBetiila. However, the presence of perforate paren-thosomes in the dolipore septa of the mycorrhizalfungus of Betula and unperforate ones in theendophyte of Cryptothallus (Ligrone et al., 1993)indicates that these are not the same.

In view of the widespread co-occurrence ofliverworts with rhizoidal ascomycetes atid Fricaceaeit is of ititerest to cotisider the possible ecological atidphysiological significance of their susceptibility toinfection by the same fungal endophyte.

Sitice the liverworts fonn permanent mats cover-

Figures 9-17.Figures 9 and 10. Erieoid myeorrhizas resynthesized from the Kurzui lungus and stained with 5 /ig ml"'DiOC|.(3) for 5 min. (a) Fluoreseenee and (b) corresponding bright lield images. Figure 9. Vaeeiniumeorymbostim, extensive development of hyphae over the root surface, x 540. Figure 10. lntracellular hyphae inepidermal cells of a hair root of Calluna vulgaris, x 500.Figures 11 and 12. Kurzia pauciflora. Figure 11. Swollen-tipped rhizoids infected with hyphae ofHvmeiioscyplius ericae, x 500. Figure 12. Uninfeeted rhizoid tips in the presence of Oidiodendron griseum,X 330. Figures 13 and 14. Calypogeia muelleraiia. Figure 13. Swollen-tipped rhizoids infected withHymenoscyplius erieae, x 240. Figure 14. Rhizoids remain uninlected in the presence of Oidiodendron mains,X 250. Figures 15 and 16. (^cpbalozia coiiniveiis. Figure 15. Swollen-tipped rhizoids infected withHymenoscyplius erieae, x 240. Figure 16. Detail of a rhizoid tip filled with hypliae of Hymeiio.<:cypliu.':, x 440.Figure 17. Ceplialozia hiciispidata; irregular rhizoid tips infected with Hymeiioseypbus ericae, x 420.

446 y. G. Duckett and D. J . Read

Figures 18-26.

Figures 18 and 19. Cephalozia bieuspidata. The rhizoids retain normal morphology and are not infeeted hyOidiodendron griseum (Fig. 18) nor by Ceratobasidium cornigerum (Fig. 19). Both, x 300. Figures 20 and 21.Cephalozia loitlesbergeri, young gametophytes frotn germinating spores showing infeetion of rhizoids andmucilage papillae (arrowed) with Hymenoscyplius ericae. Figure 20. x 190. Figure 21. x 600. Figures 22-26.Nowellia eurvifolia. Figure 22. Uninfected rhizoid. Note the pockets of mueilage along its surface and theacentrically-loeated nueleus (arrow) x 590. Figures 23—25. Stages in rhizoid infeetion with Hymenoscypliusericae. Figure 23. External hyphae, x 470. Figure 24. Numerous internal hyphae in a slightly distorted rhizoidtip, X 600. Figure 25. Hyphae in the base of a rhizoid. The other rhizoid on the left is uninfeeted, x 680.Figure 26. Wild rhizoid packed with hyphae and with an irregularly-swollen tip, x 730.

ing the surface of many acidic habitats in wetlandsand the surface of trees as epiphytes, the fact thatthey are carriers of a fungus known to be associatedin a mutualistic manner with ericaceous plants islikely to be of considerable ecological importance.The delicate hair roots of seedlings of many

ericaceous species fail to extend into acidic peatysubstrates iti the absence of infection by H. ericae(BurgefT, 1961; Read, 1991). The presence ofinoculum is therefore a prerequisite for establish-ment of seedlitigs iti tnany natural habitats.

It can be envisaged that radicles emerging from

Ericoid mycorrhizas and liverioort rhizoid-aseontycete associations 447

seeds germinating on such liverwort mats rapidlymake contact witb, and become infected by, com-patible inoculum associated with the hepaticrbizoids. Relationships of this kind between liver-worts and ericaceous sbrubs may also be importantin non-wetland sites. For example, under the densecanopy of tbe invasive shrub Rhododettdron pontictitn,which poses a threat to tbe ground flora of n-ianytemperate woodlands, among the few species toflourish are the leafy hepatics Telaranea itetitatodesand Lepidozia reptaits. Since tbese are also a potentialbost to H. ericae thev' may be inv-olvcd in facilitatingtbe invasion of native woodlands by the exotic sbrub.

It bas been revealed (Read, 1983, 1991 ; Leake &Read, 1991) that H. ericae has a range of biochemicaland physiological attributes which, when expressedunder tbe control of the ericoid plant, cai-i provide itshost with significant enhancement of nutrient avail-ability as well as resistance to tbe ad\-erse physicaland chemical impacts of acidic peaty environments.It is likely that some of these attributes are expressedto the benefit of liverwort hosts, but as yet tbefunctional significance of the association for theseplants bas not been investigated. The possibilityemerges of two-way transfer of assimilates and othernutrients between the two unrelated hosts by way oftheir shared fungal symbionts. This aspect of tberelationship together with its other functional attri-butes are currently being investigated.

A C K N O W I, l£ D G V. M !• N TS

The authors thank Professor Karen Sue Rcnzaglia forproviding the North American specimens used in thisstudy and Paul I'lctclicr for skilled technical assistance.

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Burgeff H. 1961. MiUrobiologie des 1 lochmoores. Sluttgart:Clustav l-'ischcr.

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