Ontogeny of synthesized Picea abies (L.) Karst.-Hebeloma crustuliniforme (Bull. ex St Amans) Quel. ectomycorrhizas

<̂K-- PhytoL (1992), 120, 359-369

Ontogeny of synthesized Picea abies (L,)¥i^Tst,-Hebeloma crustuliniforme (Bull, exSt Amans) Quel, ectomycorrhizas

BY I. BRUNNER AND C. S C H E I D E G G E R

Swiss Federal Institute for Forest, Snow and Landscape Research (IVSL),CH-8903 Birmensdorf, Switzerland

{Received 22 July 1991; accepted 12 November 1991)

SUMM.^RY

Ectomycorrhizas were synthesized between Picea abies (L.) Karst. and Hebeloma crustuliniforme (Bull, ex StAmans) Quel. in growth pouches containing activated charcoal filter paper. Immediately emanating hyphae frominoculated plugs contacted root hairs and root surfaces, hyphal morphology was altered. They became short-celledwith few clamp connections, highly branched, and lacked cell wall ornamentation. Then, hyphae on root surfacesformed an inner synenchymatous mantle and loose hyphae formed an outer prosenchymatous mantle..Simultaneously, root cap cells became invaded by fungal hyphae and their remnants were incorporated into theinner mantle. Further root elongation took place but root hair production was suppressed. Thirdly, a Hartig netbegan to form in basal portions of developing ectomycorrhizas and the funga! mantle became denser and thicker.Fourthly, growth of the root was reduced and the hyphae of the Hartig net penetrated between cortical ceils movingtoward the apex. Fifthly, metacutis occasionally surrounded the meristem. At this stage the formation of theectomycorrhiza can be considered to be complete and the rootlet itself is temporarily in a dormant state.

Key words: Picea abies, Hebeloma crustuliniforme, ectomycorrhizal synthesis, ontogeny, low-temperature scanningelectron microscopy.

INTRODUCTION

When studying ectomycorrhizal infection two typescan be recognized. Either an infection takes place onan uninfected root (primary infection), or new rootsurfaces become infected by inoculum already es-tablished on. or within the tissue of, older parts ofthe roots (secondary infection) (Marks & Foster,1973). Infection occurs in either case between cellsbehind the root cap and apical meristem and in frontof the region where the primary cortex becomesmoribund, a region called the mycorrhizal infectionzone (MIZ; Marks & Foster, 1973).

Early developmental stages of ectomycorrhiza]formation include first contacts of fungal hyphaewith root hairs and root surfaces, penetrationbeneath surface root cells, proliferation of the hyphaeon the root surface to form a mantle, and penetrationinto the root to form a Hartig net (Piche, Peterson &Ackerley, 1983; Piche, Peterson & Massicotte, 1988).During these processes fungal morphology may bealtered after contact with root hairs, root surfaces, andcortical cells as shown by, e.g. Jacobs, Peterson &Massicotte (1989) and Moore, Massicotte & Peterson(1989). Between the cortical cells fungal hyphae

form a labyrinthine tissue with fingerlike structurestermed palmettes (Strullu, 1979; Nylund &Unestam, 1982; Blasius et al., 1986; Kottke &Oberwinkier, 19866; Melville et al., 1987a).

Ectomycorrhizal development on short lateralroots of Picea abies (L.) Karst. seedlings can followtwo fundamentally different courses. The Hartig netis established either in advance of, or after, mantleformation (Kottke & Oberwinkler, 19866).Piloderma croceum Erikss. & Hjortst. is a represen-tative of the first type, where fungal penetrationoccurs after the formation of a loose hyphal envelope,followed by a change of morphology of Hartig netand envelope into labyrinthine tissue after a certain' incubation' time (Nylund & Unestarn, 1982).Amanita muscaria (L.: Fr.) Hooker is a represen-tative of the second type where fungal penetrationstarts only after a synenchymatous structure in themantle has differentiated (Kottke & Oberw-inkler,1986a). The time required for these processesprobably depends on the fungal species, althoughthere is little information in the literature.

The present study complete earlier descriptions ofP. abies-Hebeloma crustuliniforme (Bull, ex StAmans) Quel. ectomycorrhizas (Brunner, 1991;

360 /. Brunner and C. Scheidegger

Figures 1-4. For legend see opposite.

Ontogeny of synthesized ectomycorrhizas

Brunner, Amiet & Schneider, 1991), documents firstcontacts between fungal hyphae and lateral roots,and illustrates the stage of ectomycorrhizal de-velopment until mantle and Hartig net arecompleted.

MATERIALS AND METHODS

Seeds of Picea abies from Dielsdorf (Ct. Zurich,Switzerland) were surface sterilized for 30 min in30 and germinated on water agar plates.After 2-3 wk the seedlings could be used forsynthesis experiments. Myeelia of Hebeloma crustu-liniforme was isolated in 1990 from fruit bodiescollected in a 100-yr-old P. abies forest inMoosboden (Ct. Freiburg, Switzerland).

Autoclaved polyethylene pouches (Cellpack,Switzerland) 13x16 cm with an activated charcoalfilter paper (Schleicher & Schuell, Switzerland) werefilled with 10 ml modified Melin-Norkrans nutrientsolution (MMN: Marx & Br>an, 1975), withoutglucose and malt, and one seedling was inserted. pHwas 65. After 2-3 months, when the root systemswere developed, 4—6 inoculum discs were placed inthe vicinity (max. 3 mm) of the lateral roots andanother 10 ml MMN (including 5 g 1"̂ glucose, nomalt) was added. Fungal discs were grown prior toinoculation on solid MMN-media including glucose(10 gl" ') and malt (3g l - ' ) for 5 d. Two strips offoam were introduced to provide air space in thepouch. Sterile distilled water was added frequently.The synthesis experiments were carried out in agrowth chamber with a 16h day period (PAR:100 piTno\ m"^ s" )̂ at 20 °C and 70 % humidity.

Two seedlings were harvested every few days andshort roots were fixed in 25 "o glutaraldehyde. Fourto six short roots were dehydrated in alcohol andembedded in glycol-methacrylate. Two specimens(or more if necessary) were longitudinally sectioned

361(1-5//m), and stained with Giemsa for chitinaceousmaterial (Clark, 1981, p. 267). Micrographs weretaken with a Leitz Aristoplan photomicroscope.

For low-temperature scanning electron micro-scopy (LTSEM), 4—6 charcoal filter paper discsincluding ectomycorrhizal lateral roots of variousdevelopmental stages were cut, mounted on alu-minium stubs with 1 "o methylcellulose (MethocelMC, medium viscosity, Fluka) in H2O, and frozen inliquid nitrogen immediately thereafter. The speci-mens were then transferred onto the cold stage in thepreparation chamber of a Balzers SCU 020 unit(Muller ei al., 1991 ; Scheidegger et al, 1991) andpartially freeze-dried for 10 min at — 80 °C in a highvacuum (P < 2 x 10"'* Pa). Gold sputter coating wasthen carried out after raising tbe pressure to 2 2 Pa.The coating thickness was 15 nm, measured by aquartz thin film monitor. After coating, thespecimens were transferred with a manipulatorthrough the sliding vacuum valve onto the SEM coldstage in a Philips SEM 515. The temperature on thecold stage in the microscope was kept below— 120°C. After observation, the specimens werewithdrawn onto the cold stage in the preparationchamber, fractured with a microtome and partiallyfreeze-dried at —90°C for 1 min., sputter coatedas described above, and observed again in themicroscope.

RESULTS

Development of the ectomycorrhizas

Picea abies seedlings in growth pouches on activatedcharcoal filter paper form an extensive root systemwith many first order lateral roots within 2-3 months(Fig. 1). Hyphae emanating from the fungal plugscontact root hairs and root surfaces, and they induceectomycorrhizas on lateral roots as soon as 4 d afterinoculation. Hyphae form a weft and grow along

Figures 1—4. Seedlings of Picea abies inoculated with Hebeloma crustuliniforme.

Figure 1. Seedling in a growth pouch, 47 d after inoculation with mycelial plugs of H. crustuliniforme.Numerous ectomycorrhizal lateral roots (arrowheads) have formed. Scale bar = 1 cm. Figure 2. Matureectomycorrhizal lateral roots 48 d after inoculation. Hyphae have enveloped the roots and formed a mantle.Lateral roots are interconnected by a weft of hyphae (arrowheads) following the main root axis. Scale bar— 1 mm. Figure 3. Low-temperature scanning electron micrograph of a young ectomycorrhizal lateral root10 d after inoculation. Hyphae (arrowheads) contact and interact with root hairs and root surface in the apicalregion. Numerous root hairs (double arrowhead) are evident. Scale bar = 0-3 mm. Figure 4, Low-temperaturescanning electron micrograph of a mature ectomycorrhizal lateral root 71 d after inoculation. Root hairs areabsent on new lateral roots from the outset and surfaces are covered with compact mantles which are connectedby looseiy interwoven hyphae formmg a weft (arrowheads). Scale bar = (MS mm.

Key to abbreviations used on Figures 5 et seq.

AM apical meristem OM outer mantle

CC cortical cells RC root cap cells

IM inner mantle

RD rhizodermal cells

RH root hairs



Ontogeny of synthesized ectomycorrhizas 363

main roots to colonize distant lateral roots (Figs 1,2). Mature ectomycorrhizas can be observed at theearhest 6-7 wk after inoculation. Ectomyorrhizas arecharacterized by a white mantle, covered with whiteinterwoven hyphae forming a hyphal weft (Fig. 2)which occasionally interconnects ectomycorrhizalroots (Fig. 4).

Low-temperature scanning electron microscopy

Hyphae from mycelial plus contact root hairs androot surfaces on the apical portion of the lateral rootswithin 2 wk of inoculation (Fig. 3). In the followingweeks, hyphae further envelope the whole lateralroot in a very loose manner and form a loose and thinmantle either at the apex or along the length of theroot. Root hairs at the basal portion of the lateral rootcollapse. Growth of ectomycorrhizal roots slowsdown 4—7 wk after inoculation when they areapproximately 1-2 mm long. Formation of root hairson new lateral roots is suppressed from the outsetand a fungal mantle is formed immediately after theappearance of the lateral root (Fig. 4).

Striking morphological changes occur in thehyphae that contact root surfaces. Aerial hyphae arestraight, rarely branched, long-celled with frequentclamp connections, and ornamented, while those inclose contact with the surface of root hairs (Fig. 5)and roots (Figs 6, 7) are often bent, highly branched,short-celled with few clamp connections, andsmooth. The absence of ornamentation on the outerhyphal wall is strongly dependent on contact withroot surfaces (Fig. 8).

Young ectomycorrhizas have a differentiatedhyphal mantle with a prosenchymatous outer and asynenchymatous inner layer, although a Hartig nethas not yet developed (Fig. 9). Mature ecto-mycorrhizas (Fig. 10) differ from young ecto-mycorrhizas mainly by the presence of a Hartig net.Hyphal morphology is distinctly different in theouter and inner mantles. While outer mantle hyphaestill can be recognized as individual long-celled andrarely branched hyphae, inner mantle hyphae areshort-celled and highly branched giving a ladder-likeappearance (Fig. 11) similar to that observed in theHartig net. In addition, hyphae of the inner layer areembedded in a matrix (Fig. 11).

Light microscopy

Longitudinal sections through non-ectomycorrhizallateral roots show that the roots possess an apicalroot cap and intact root hairs in the subapical region(Fig. 12). Root cap cells can be observed up to thesubapical region and the apical meristem is distinct.Young ectomycorrhizal roots are loosely envelopedby a thin two-layered mantle (Fig. 13). Root cap cellwalls begin to dissolve or are damaged and fungalhyphae start to invade root cap cells. Root hairsoccasionally collapse and root hair formation isabsent at the growing apical portion. The apicalmeristem is distinct and chromosomes can be seen atvarious mitotic stages, indicating cellular division(Fig. 13). The Hartig net is not obvious at this stageof development. Pre-inature ectomycorrhizal rootshave a thickened two-layered mantle (Fig, 14). Rootcap cells are mostly fragmented and their cell wallshave been incorporated in the inner mantle. Theapical meristem is smaller than in young ecto-mycorrhizas, indicating a reduction of root growth(Fig. 14). A Hartig net is visible in the subapicalportion of the root indicating that invasion of fungalhyphae between cortical cells progresses through thecortex towards the root apex (Fig. 14). It eventuallyreaches the apical portion behind the nieristem whenroot growth has slowed down. Mature ectomycor-rhizas have a two-layered fungal mantle and a Hartignet reaching to the endodermis and as far as themeristematic zone (Fig. 15). The apical meristemcan be surrounded by a metacutis indicating that celldivision has ceased. Root cap cells and root hairs arenot in evidence in mature ectomycorrhizas (Fig. 15).

After contacting the young lateral root, fungalhyphae enter a mucilaginous layer on the surface andform a loose synenchymatous layer (Fig. 16). Afterthat, they penetrate between root cap cells (Fig. 16)and into damaged root cap cells (Fig. 17), or rarelyinto intact root cap cells (Fig. 18). After invasion ofthe root cap cells (Fig. 19), the remaining cell wallsbecome incorporated into the inner fungal mantle(Fig. 20). Although no Hartig net has yet developed,the fungal mantle is composed of two distinct layers(Fig. 21).

Figures 5-8. Low-temperature scanning electron micrographs of altered fungal morphogenesis duringinteractions between hyphae of Hebeloma crustuliniforme and root hairs and root surfaces of Picea abies.

Figure S. Hyphae branch (arrowheads) in close contact with the root hair surface. Straight, ornamented, andsparsely branched hyphae are present (double arrowhead) but not in contact with root hairs. Scale bar = 10 /fni.Figure 6. Hypbae branch (arrowheads) in close contact with the root surface. Straight and sparsely branchedhyphae are present above the root surface (double arrowhead). Scale bar = 10/^m. Figure 7. Close-up ofhyphae which contact the root surface. Hyphae become short and extensively branched with somewhat swollenand fused appearance (arrowheads). Scale bar — 10//m. Figure 8. Close-up of hyphae which contact a roothair. Hyphae do not bave ornamentation in the contact zone (arrowheads). Scale bar = 5 /im.


11Figures 9-11. Low-temperature scanning electron micrographs of longitudinal fractures of Picea abies-Hebeloma crustuliniforme ectomycorrhiza] roots at various stages.

Figure 9. Young ectomycorrhiza! lateral root 10 d after inoculation. An inner and outer mantle have formedbefore hyphae have penetrated into the root. Scale bar = 10//-m. Figure 10. Mature ectomycorrhizal lateralroot 46 d after inoculation. An inner and outer mantle and a Hartig net (arrowheads) between rhizodermal celisand cortical cells are present. Scale bar — 10 jUm. Figure 11. Mantle of a mature ectomycorrhiza! lateral root71 d after inoculation. The inner mantle is embedded in a matrix (arrowheads). Scale bar = 5 //m.



366 J. Brunner and C. Scheidegger

DISCUSSION

The most striking advantages of the growth pouchtechnique are the direct obser\'ation of the ecto-mycorrhizal development and the possibility ofharvesting the ectomycorrhizal roots without majordisturbance of the mantle surfaces (Fortin, Piche &Laionde, 1980). The possibility of autoclaving theplastic bags prolongs the experimental period andeven slow-growing conifers such as Picea can beused. In addition, activated charcoal filter papers,first used by Kottke et al. (1987), absorb noxiousplant and fungus exudates which might inhibit rootgrowth. Recent investigations on the ontogeny andstructure of angiospermous ectomycorrhizas such asthose observed in Alnus (e.g. Massicotte, Peterson &Melville, 1989; Brunner, Brunner & Miller, 1990),Betula (e.g. Massicotte et al., 1990), Eucalyptus (e.g.Moore et ai, 1989; Jacobs et al., 1989), and Dryas(e.g. Melville, Massicotte & Peterson, 19876) haveemployed this technique. In addition, the use of thecryo-preservation technique allows the examinationof root surfaces as well as inner structures of rootsexposed by fractures in fully hydrated conditions.Collapse of root hairs induced by the freeze-dryingor shrivelling induced by critical-point drying can beavoided (Sargent, 1986). This technique was firstapplied to ectomycorrhizas by Alexander, Jones &McHardy (1987) who compared it with criticalpoint-drying.

The ontogeny of P. abies-Hebeloma crustuliniformeectomycorrhizas mainly follows the steps proposedby Kottke & Oberwinkler (1986a) for P. abies-Amanita muscaria ectomycorrhizas. Fungal penetra-tion into the cortex only takes place after thesynenchymatous structure of the mantle has beendifferentiated. A synenchymatous inner and looseprosenchymatous outer layer of the mantle wasobserved as early as eight days after inoculation withfungal plugs. During that time, root cap cells becameinvaded or penetrated by fungal hyphae, a processwhich is not identical with Hartig net forn^ation.Remnants of root cap cells, mainly cell walls, becameincorporated into the inner layer of the mantle. Sucha process has been observed by several other authors,e.g. Marks & Foster (1973), Duddridge & Read(1984), Kottke & Oberwinkler (1988), Horan,Chilvers & Lapeyrie (1988), Moore et al. (1989).

The morphological similarity between hyphae of

the inner mantle and the Hartig net is obvious.Jacobs et al. (1989) suggested a model which treatsthe inner mantle and the Hartig net as relatedcomplexes. Both are in close contact with root cellsand thus the change of hyphal morphology mighthave been induced by root exudates. That bothorgans fulfil similar physiological functions withinfunctioning symbioses has been proposed (Jacobset al., 1989) but also criticized by one reviewer of thatpaper. The Hartig net is believed to play the mostimportant role in transporting and exchangingnutrients due to its transfer-cell-like organization(Kottke & Oberwinkler, 1987). Also it is believedthat hyphae of the Hartig net grow in a definitedirection, transverse to the root axis, from therhizodermis to the endodermis, and not randomly(Kottke & Oberwinkler, 19866), as is the case of thehyphae of the inner mantle (Warmbrodt & Eschrich,1985).

The general feature used to confirm the ectomycor-rhizal status of a root is the presence of a Hartig net(Harley & Smith, 1983). In the present study, aHartig net was never established without the pres-ence of an outer and inner mantle on the root surface.Hartig nets were located only in the basal portion inyounger ectomycorrhizas but m the whole cortex,reaching the apical meristem, in older ectomycor-rhizal roots. It appears that the first intercellularpenetrations of hyphae to form a Hartig net occur {a)in a zone behind the apex in the so-calledectomycorrhizal infection zone, and (b) only fromthe inner mantle and only after a delay. Such a delaynnight be explained by the physical barrier of thepectic middle lamella between the cortical cell walls,since the fungal hyphae have to split it apart bymechanical forces (Nylund & Unestam, 1982).During that delay, the lateral root grows on and thusa Hartig net can be observed firstly in the basalportion only and never immediately behind theapical meristem. According to Horan et al. (1988),only new root cells, formed after fungal invasion ofthe root cap tissue, are involved in the process ofHartig net formation. Hyphae of the Hartig netreach the apical portion probably only when therootlet has retarded its growth. Simultaneously, areduced apical meristem is present. A metacutislayer around the apical meristem then mdicates apossible conclusion to the formation of the ecto-mycorrhiza. A similar process of ectomycorrhizal

Figures 12-14. Light micrographs of longitudinal sections of Picea abies-Hebeloma crustuliniformeectomycorrhizal roots at various stages.

Figure 12. Non-ectomycorrhizal lateral root. Root hairs and root cap cells are evident. Scale bar — 0*1 mm.Figure 13. Young ectomycorrhizal lateral root 16 d after inoculation. Hyphae form a loose prosenchj'matnusouter mantle and a synenchymatous inner mantle (arrowheads) in contact with the root surface. A Hartig netis not visible, although hyphae penetrate between root cap cells and rhizodermal cells (double arrowhead). Scalebar = 0-1 mm. Figure 14. Pre-mature ectomycorrhizal lateral root 32 d after inoculation. A Hartig net(arrowheads) is developing in the basal portion of the root. Scale bar = 0-1 mm.



368 / . Brunner and C. Scheidegger

formation has been demonstrated by Kottke &Oberwinkler (1986a) for P. abies-A. muscaria and byMelville et al. (1987 b) for Dryas integrifolia(V ah\.)~Hebeloma cylindrosporum Romagn. ecto-mycorrhizas. In Dryas, a decrease in root elongationbefore infection by the ectomycorrhizal fungus wasnot detected.

In conclusion, the process of ectomycorrhizaformation of the present system P. abies-H. crustu-liniforme synthesized with the growth pouch tech-nique can be demonstrated with the followingapproximate timetable. In the first week, hyphaefrom inoculated plugs contact root hairs and rootsurfaces, and immediately hyphal morphology isaltered. They become short-celled with few clampconnections, they are highly branched, and they lackornamentation of the cell wall. In the second andthird week hyphae form an inner synenchynriatouslayer at the root surface. A loose hyphal envelopeforms around the lateral root. Both layers togethercan be designated as the fungal mantle of thedeveloping ectomycorrhiza. At the same time, rootcap cells start to break dow n or their cell walls becomedamaged. They are invaded and penetrated by fungalhyphae, and their cell wall debris is incorporatedinto the inner mantle. In the course of rootelongation, formation of root hairs is suppressed andexisting root hairs collapse. In the fourth week, thefirst hyphae of the Hartig net can be observed inbasal portions of developing ectomycorrhiza. Duringthat process and in tbe following week, fungalmantles become denser and thicker. In addition, rootgrowth is reduced and thus hyphae of the Hartig netinvade the whole cortex of the ectomycorrbizalinfection zone up to the apical meristem. In the sixthweek the first metacutis layers in ectomycorrhizalroots can be observed. The process described differssignificantly in one main point from that describedby Nylund & Unestam (1982). In their system ahyphal envelope, which is, according to them notidentical with a fungal mantle, develops after firstcontact between the fungus and the root. Mantle

formation occurs only after the Hartig net hasdeveloped and fungal byphae change their mor-phology after a certain incubation time within theroot cortex. Obviously, it is possible that fungalhyphae do not change their morphology immediatelythey have contacted a compatible root. However,Piloderma croceum so far seems to be the only closelyinvestigated fungus compatible with Picea thatchanges its morphology only after a certain in-cubation time.

ACKNOWLEDGEMENTS

We thank Mrs B. Schneider for sectioning and staining theectomycorrhizas, Mrs M. ZoHinger for assistance in syn-thesizing the material, Mr P. Hatvani for assistance at theLTSEM, and Mrs M. J. Sieber for correcting the Englishtext.

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Figure 15. Mature ectomycorrhiza] lateral root 36 d after inoculation. The Hartig net (arrowheads) isdeveloped and reaches the metacutis (double arrowhead) which surrounds the apical nneristem. Scalebar — O'l mm. Figure 16. Young ectomycorrhizal lateral root 8 d after inoculation. Fungal hyphae (arrowhead)are located in a mucilaginous layer on the surface of the root and between root hairs and penetrate betweenroot cap cells parallel to the root axis (double arrowheads). Scale bar =10 fim. Figure 17. Young ectomycorrhizallateral root 8 d aftet inoculation. Fungal hyphae have penetrated into intact (arrowhead) and damaged (doublearrowhead) root cap cells. Scale bar = 10//m. Figure 18. Young ectomycorrhizal lateral root 8d afterinoculation. A fungal hypha (arrowhead) from the inner mantle has penetrated into a root cap cell. Scalebar =10 //m. Figure 19. Young ectomycorrhizal lateral root 16 d after inoculation. Fungai hyphae(arrowheads) have penetrated between root cap cells and rhizodermal cells. Scale bar = 10/im. Figure 20.Young ectomycorrhizal lateral root 16 d after inoculation. Inner mantle incorporating cell walls from the root capcells (arrowheads). Scale bar = 10//.m. Figure 21. Young ectomycorrhizal lateral root 16 d after inoculation.Fungal hyphae form a double-layered mantle with a loose prosenchytnatous outer and in contact with the rootsurface a synenchymatous inner mantle. Scale bar = 10/zm.


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