Ericoid Mycorrhizas and Rhizoidascomycete Associations

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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 vitroBY 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)

SUMMARYThe hypothes is 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 bygrowing the l iverworts withpure cul tures of the putative aseomycetous ericoid endophyte Oidiodendron, with the basidiomycetous endophyteof orchids, Ceratobasidiiim cornigeriiiii, and with several ectomyeorrhizal fungi.

It was confirmed that them e m h e r s of these li\-erwort families tested, most of which are associated in nature withericaceous plants, were readily infected byH. ericae, as well as hy isolates obtained from the l iverworts 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 northeorehid or eetomyeorrhizal fungi infected thel iverworts, and all failed to reproduce the characteristic swelling of the rhizoid tips which is seen in nature andin plants inoculated with H. ericae.Th e resul ts provide amajor extension of the likely host range o(H. ericae and may liave eonsiderahle significancefor hoth groups of plants which, as a consequence of shar ing the same endophyte , have the potential to act as

sourees of inoculum for each other in na ture . 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.

I N T K O D U C T I O N to, t l i o s e r e p o r t e d to f o r m e r i c o i d m y c o r r h i z a in thhair roots of major ericaceous genera such as Callun

I'he widespread occurrence of aseomycetous fungi Erica, Rhododendron and Vaecinium. In additionin the rhizoids of leat'y liverworts of the families the structural and ultrastructural similarities beLepidoziaceae, Calypogeiaeeae, Adelanthaceae, tween the hyphal coils formed in the swollen rhizoiCephaloziaceae and Cephaloziellaeeae has been con- apices and flagellifortn axes of the leafy hepatics ati


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440 y.G. Duckett and D . J. R eadmountains (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 toexamine 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, aspointed out by Leake &Read (1991), is one of the major prerequisites for thesatisfaction of Koch's postulates, has previouslyenabled confirmation of the ability ofH. ericae toform ericoid associations in Galluna (Pearson &Read, 1973 ; Bonfante-Easolo & Gianinazzi-Pearson,1982), Erica (Duclos, Pepin & Bruchet 1983; Read,1983), Rhododendron (Du ddridg e & 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 , Tsune da & Murakami1993).

Considerable specificity in the relationship be-tween H. ericae and its host plants issuggested 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 (Giovane tti &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 METHODSAxenic cultures of a variety of liverworts (Tables 1and 2) were established from spores, gametophyteapices or gemmae on Parker agar (Klekowski, 1969)

Stems ofCalypogeia jissa and subterranean aof Kurzia paueijiora, Cephalozia bieuspidata,leuca?ttha, Odontoschisma deniidattitn and O. sbearitig tiumerous fungus-containing rhizoids weseparated trotn their native substratum by thorouwashing in distilled water and any adherent organmatter was i-emoved with a fine paint brusEollowing 5 s immersion in hypochlorite anseveral rinses in sterile distilled water the axes weplanted on 2 "o malt or tiutrient-free agar at rootemperature. Hyphae grew out from the rhizoiwithiti a ten day period on both media. They wesubcultured, and subsequently used to inoculate taxenic liverwort cultures or axenic seedlitigs of Erieinerea, Calluna vidgaris and Vaecinium corymbL. which had been established from surfacsterilized seeds.Axenic ericaceous seedlings and samples of taxenically cultured liverworts were also addedagar cultures of the following known mycorrhizfungi: Hymenoscyphus ericae (Read) Korf. & K er(isolates 100 atid 101 from Calluna vulgaris) Oiddendroft griseum Robak (from Vaecinium corymbosum), O. maius Baron and Cenococcum geophEr. [both from Picea sitchensis (Bong.) Carr], Suiboviniis (Er.) O. Kuntze and Anianita musearia (LEr.) Llooker (both from Pinus syhestris L.) Paxilltinvolittus (Er.) Er. (from Betula pubescens Ehrh.)Ceratobasidium eornigertun Rogers [from Goodrepens (L.) R. Br.]. Eull details of these isolates given in Duckett & Read (1991).Cleaned specimens of the following liverwortCephalozia hiberniea, C. lunuUfolia, Cladopodijhiitans, Kurzia sylvatiea, K. trichoclados, Odtoschisma prostratum, Mylia atiomala and Telarnematodes, which typically contaiti rhizoidal asmycetes (Table 1) were placed in Petri dishcontaining either distilled water, Parker nutriesolution or damp silica sand and the culturmaintained over periods of several months under thlaboratory cond itions detailed above. All new growwas fungus-free (Duckett et al., 1991). At regulinter\'als these cultured plants plus those of taxenically-grown liverworts were returned to theorigitial substrata or foreign natural substrata ('liab1) maintaitied in the laboratory in plastic boxlightly covered with polythene, and their furthdevelopment monitored.Living specimens were mounted in water anobserved and photographed under differential interferetice contrast optics with a I^eitz Ortholumicroscope. Some of the materials were also stainein 5 //g m L' 3,3'-dihexyloxacarbocyanine iodi


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Ericoidmveorrliizas and liverzvort rhizoid-ascomvcete associations 44Sourees of liverworts forming rhizoid-ascomycete associations

Original locality and substrate Hepatics present Ericaceae presentbog peat, Cranesmoor, New

Forest, England

hog peat, Woodfield, CentralIreland

Sandstone rocks, E. Susse.x. lingland

) Qiierciis woodland, Co. Kerry, Westot Ireland

Rotten logs, Merioneth, N. Wales

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

Peaty soil, heathland f'ast Anglia,England

Peaty soil, ravine Merioneth N.Wales

Rock faee. Red l


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Ericoid mvcorrhizas and liverivort rhizoid-ascomvcete associations 44

Figures 1-8.F igu res 1 and 2 . Kurzia pauciflora. Hy pha e growing into water agar from the swollen t ips of infected rhizoids,x 4 1 0 . Figure 3 . Erica cinerea, t ip of a wild hair root showing fungal hyphae in the mucigel; cells of the


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J. G. Duckett and D. J. Read


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Ericoid mycorrhizas and liverivort rhizoid-ascomycete associations 445presetice of a variety of fungal isolates. Pictures ofnaturally-occurritig futigus-infected rhizoids of thesame species may be found in Duckett et al. (1991)and D ucke tt & Read (1991). W hereas 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 l iverworts 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. Init ia l ly the hyphae growaround the surface of the rhizoids (Fig. 23) mostlikely within the superficial mucilage pockets (Fig.22). Subsequeti t ly penetrat ion 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 me dullary cells of the hepatic s tem s.

The results of all the resynthesis experiments aresum mariz ed in Ta ble 2. T h e 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 substra ta (Ta ble 1) their new rh izoidsdeveloped asconiycete associatiotis itidistinguishablefrom those found originally or resynthesized in

culture. Part icularly notable am ong these results wathe formatioti of rhizoid-futigus associations in: (1Odontoschisma prostratum, Calvpogeia nutelleranaatid Kttrzia sylvatiea of North Americati origiti otBritish substrata; (2) the bog species Mylia anomalaand Cladopodiella fluitans oti rotteti logs and soifrom beneath Rhododendrons far removed from theinatural habitat and (3) Noivellia curvifolia, tiormallya rotteti log species, on terrestrial peaty soil.DISCUSSIONThe results provide confirmatioti of the hypothesithat the rhizoids of a range of leafy liverwort specieof the fatiiilies Lepidoziaceae, CalypogeiaeeaeCephaloziaceae and Cephaloziellaeeae cati be itifected by the ericoid mycorrhizal fungus Hymenoscyphus ericae, which produces in their swolletapices hyphal coils typical of those seen in natureThey also provide a very considerable increase in thlikely host range of this fungus. An apparentlysitnilar ascotnycete futigus has been isolated fromrhizoids of Ceplialoziella exiiflora growing in Antarctica (Williams, Roser & Sep pelt, 1994).

As far as we are aware, the present study is the firsreport of the same fungus infectitig both vasculaatid non-vascular plants. This discovery clearlyinvites in\ 'estigation into the possibility that sotne othe zygomycetous and basidiomycetous fungassociated with other hepatics (Boullard, 1988Ligrotie & Lopes , 1989 ; Ligrotie , Pocock & Duckett1993) may be, respectively, VA and ectomycorrhiza(or orchid) mycorrhizal fungi of higher plants. Aflrst sight one highly promising pairing is that of thparasi t ic achlorophyllous hepatic Cryptothalliiinirabilis growing in close proximity to the roots oBetiila. However, the presence of perforate parenthosomes in the dolipore septa of the mycorrhizafungus of Betula and unperforate ones in theendophyte of Cryptothallus (Ligrone et al., 1993indicates that these are not the same.

In view of the widespread co-occurrence oliverworts with rhizoidal ascomycetes atid Fricaceaeit is of ititerest to cotisider the possible ecological atidphysiological significance of their susceptibility toinfection by the same fungal endophyte.Sit ice the l iverworts fonn permanent mats cover

Figures 9-17.F ig u re s 9 a nd 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. Fi gu re 9. Vaeeiniumeorymbostim, extensive development of hyphae over the root surface, x 540. F ig u re 10. lntrace llular hyphae inepidermal cells of a hair root of Calluna vulgaris, x 500.Figu res 11 an d 12. Kurzia pauciflora. Fig ur e 11. Swo llen-tipped rhizoids infected with hyphae of


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

Figures 18-26.F i g u r e s 18 and 19. Cephalozia bieuspidata. The rhizoids re ta in normal morphology and are not infeeted hyOidiodendron griseum (Fig. 18) nor by Ceratobasidium cornigerum (Fig. 19). Both , x 300. F i g u r e s 20 and 21.Cephalozia loitlesbergeri, young gametophytes f rotn germinat ing spores showing infee t ion of rhizoids andmucilage papillae (arrowed) with Hymenoscyplius ericae. F i g u r e 20. x 190. Figure 21 . x 600. Figures 22 -26 .Nowellia eurvifolia. F i g u r e 22. Uninfec ted rhizoid. Note the pockets of mueilage along its surface and theacentrically-loeated nueleus (arrow) x 590. Figures 2325. Stages in rhizoid infeetion with Hymenoscypliusericae. Figure 23 . External hyphae , x 470. Figure 24 . Numerous interna l hyphae in a slightly distorted rhizoidt ip, X 600. F i g u r e 25. H y p h a e in the base of a rhizoid. The other rhizoid on the left is uninfee ted, x 680.F i g u r e 26. Wild rhizoid packed with hyphae and with an irregularly-swollen tip, x 730.


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Ericoid mycorrhizas and liverioort rhizoid-aseontycete associations 44seeds germinating on such l iverwort mats rapidlymake contact witb, and become infected by, com-patible inoculum associated with the hepaticrbizoids. Relationships of this kind between l iver-worts and ericaceous sbrubs may also be importantin non-wetland si tes. 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 woo dlands by the exotic s bru b.It bas b een revealed (Read , 1983, 1991 ; Leake &Read, 1991) that H. ericae has a range of biochemicaland physiological a t tr ibutes 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 possibil i tyemerges of two-way transfer of assimilates and othernutrients between the two unrelated hosts by way oftheir shared fungal symbionts. This aspect of tberelat ionship together with i ts other functional a t tr i-butes are currently being investigated.A C K N O W I , l D G V. M ! N T SThe authors thank Professor Karen Sue Rcnzaglia forproviding the North American specimens used in thisstudy and Paul I'lctclicr for skilled technical assistance.R li I'- K R E N C E SBonfa nte -Fasolo P , Gia n ina zz i -P ear son V. 1982. I ' l i n i -strucliiral aspects ot cndoiiiyct)i-i-hizii m the l^ncaccsie. 111.Morphology of the dissociated symbionts and modificationsoccurring during their reassociation in a.xenic culture. Nexv

PhyltilogisI 9 1 : 691-704.Boullard B. 1988. Obserx-ations on the coe\-ohition ot tungi withhepatics. In; FirozynsUi KA, Hawksworth DL, eds. Coevotutiontij Fungi 7cilh Pltints antl A nimals. London: Academic Press ,l()7-124.Burgeff H. 1961. MiUrobiologie des 1 lochmoores . S lu t tgar t :Clustav l-'ischcr.Currah RS, Tsu ned a A, M ura kam i S . 1993. Conidiogenesis inOidiodendron perieonitiides and ultrastructure ot ericoid n-iycor-rhizas formed with Rhtidtitleiitlron brachyctirpum. CanadittiiJtmrnal tif Botany 71: 1481-14S.S.Dalpe Y. 1986. Axenic synthesis ot ericoid mvcorrhiza inVacciiiium angustifoliiim Ait. bv- Oidiotlendron species. NeivPhylologist 103: 391-.396.Dalpe Y. 1991. Statut endomyeorhizien du genre Oidindentlrtin.Ctiiiadian Journal of Botany 6 9 : I 71 2-1 714.D o u g l a s C G , H e s l i n M C , R e a d C. 1989. Isolation ot Oiditi-dentirtin intiius Irom Rhodotleiulrnn and ui t ras tructural charac-

terisation of synthesised mycorrhizas. Ctrnatlian Jtiurntjl Botany 67: 2206-2212.Duckett JG, Read DJ. 1991. T he tise o tt he fluorescent dye, 3,3dihexyloxacarboeyanine iodide, tor selective staining ot ascmycete tungi associated with liverwort rhizoids and ericomyeorrhizal roots. Neic Phytoltigist 118: 259-272.Ducket t JG, Renzagl ia KS, Pe l l K. 1991. A light and electrmicroscope study ot rhizoid-aseomyeete associations antlagellitorm axes in Iiritish hepatics. New PliytologisI 11233-257.Duclos JL, Pep in R , Bruche t G. 1983. Ktude niorphologiquanatomique el u l t ras truclurale d 'endomycorhizes synthet iqud'Erica ctniiea. (\intittiaii Journtd tif Bottiny 6 1 : 466MS.Dud dridg e JA, Rea d DJ. 1982. .Xn ultrastructural analysis ottdevelopment o t myeorrhizas in Rhododendron poiitieiiCanadian Journal nf Botany 60: 2345-2356.Gianinazz i -Pearson V. 1985. Host tungus speciiieity, reconition and compatibility in n-iycorrhizae. In: Verma DPHoh-i Til, eds. Plant gene research-genes involved in inicrtihpltiiil intertutitins. Berhn : Sp r inger-Ver lag , 225 -253 .Giovannetti M, Lioi L. 1990. The mycorrhizal s tatus t>( .4rbuiinedo in relation to compatible and ineompatible tung('aiiadian Journal tif Btitany 6 8 : 1 239-1244 .Klekowski EJ. 1969. The reproduct ive ecology ot t

Pteridophyta. HI. . \ s tudy ot the Blechnaceae. BtitanicalJournof the Linnean Soeiety Botany: 62, 361-377.Leake JR, Read DJ. 1991. Rxperiments w ith ericoid mycorrhizIn: Xorris JR, Read DJ, Varma .^K, eds . Methods microbiology 2.i. London: .Aeademic Press , 435-459.Ligrone R, Lopes C. 1989. Cytology and developm ent ot n-iycorrhiza-like inteetion in the gametophyte of Cnnocephalueonieum L. Dum. (Marchantiales , Hepatophyta). AVn' Phtologist 111: 423-433 .Ligrone R , Pocock K, Ducke t t JG. 1993. . \ comparat ivultrastructural analysis ot endophytic basidiomyeetes in thparasitic aehlorophyllous hepatic Cryptothallus mirabilis anthe closely allied photosynthetic species Aneura pingu(Metzgeriales) . Canadian Journal tif Botany 7 1 : 666-679.Pea rson V, Re ad DJ. 1973. The biology ot myeorrhiza in thRricaceae. I. The isolation ot the endophyte and synthesis myeorrhizas in aseptic cultures. Neie Phytoltigist 7 2: 371-37 'P o c o c k K , D u c k e t t JG , G r o U e R, M o h a m e d M A H , P a n g W C1984. Branched and swollen rhizoids in hepatics from montanrain forest in Peninsular Malaya. Journal of Bryology 1241-246.Read DJ. 1983. The biology ot mycorrhiza in the IsricaleCanadian Journal tif Botany 61 : 985 -1004 .Read DJ. 1991. Mycorrhizas in ecosystems. Experieiitia 47376 391.Schus ter RM. 1984. Kvolution, phylogeny and elassitieation the Hepaticae. In : Schuster RM, ed . New manual nf brytiltigyNiehinan; Hattori Botanieal Laboratory , 892-1092.Smith AJE. 1990. The liverworts tif Britain and IrelanCam bridge & London : Cam bridge LInivers i ty Press .Stribley DP, Read DJ. 1974. The biology ot mycorrhiza in thiM-icaceae. \\. 'I'he efieets ot mycorrhizal inteetion on thuptake ot '-'N from labelled soil by I'ticciniuin iiiacrticarpoii .ANew Phyttilogist 73: 1149-1150.Stribley DP, Read DJ. 1976. The biology of myeorrhiza in thI'^rieaeeae. VI. The effects of mycorrhizal inteetioi-i anconeei-itration ot amt-i-ionium nitrogen on growth ot eranberrv(J'aecinium maertiearptiii .-Xit.) in sand etilture. New Phytologis77: 6.1 72.Stribley DP, Read DJ. 1980. I^he biology ot mycorrhiza in thlirieaeeae. Vll. The relationship between myeorrhizal inteetioand the capacity to utilize simple and complex organic nitrogesources. Nen- Phyttilogist 86: 365 371.Wil l iams PG, Roser DJ, Seppe l t RD. 1994. Myeorrhizas ohepatics in continental .Antarctica. Mycological Research 9834-36 .


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Ericoid Mycorrhizas and Rhizoidascomycete Associations

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