Trans. Br. mycol. Soc. 73 (1) 117-125 (1979)

[ 117 ]

Printedin Great Britain

SHEATHING MYCORRHIZAS OF NURSERY GROWNPICEA SITCHENSIS

By G. W. THOMAS AND R. M. JACKSON

Department of Microbiology, University of Surrey, Guildford, Surrey GU2 5XH

Seven types of sheathing mycorrhizas of nursery-grown Picea sitchensis (Bong.) Carr. aredescribed. Four of these are formed by basidiomycete fungi of which two have been identifiedas Thelephora terrestris (Ehrh.) Fr. and Laccaria laccata (Scop. ex Fr.) Cooke. Isolatesobtained from the mycorrhizas have been used to synthesize mycorrhizas in asepticconditions.

One of the fundamental problems in the study ofsheathing mycorrhizas is the identification of thecomposite organ formed in the association.Ideally the identity of both tree and fungus mustbe determined and when this is impracticable,definition must be based upon recognition ofstable features. Trappe (1967a) suggested con-stant features of the fungus such as the presence orabsence and characteristics of clamp connexionsand rhizomorphs, and the structure and morpho-logy of the fungal sheath and associated hyphae.Dominik (1959,1969) used microscopic character-istics in an attempt to group together sheathingmycorrhizas formed by the same fungus withdifferent host species. Mycorrhizas formed by onespecies of fungus may vary in gross morphologydepending upon the host plant, but the mantles,attached mycelium and rhizomorphs remain verysimilar (Zak, 1973).

Identification of the fungal partner has usuallybeen based on observation and identification ofsporophores in the vicinity of mycorrhizas. Amore satisfactory method is by direct isolation ofthe fungus from the mycorrhiza and comparisonwith known cultures. Techniques for isolationfrom sheathing mycorrhizas have been developedby Zak & Bryan (1963), Zak & Marx (1964) andMarx, Bryan & Grand (1970). Unfortunatelyidentification based upon isolation is limited bythe difficulty of culturing mycorrhizal fungi andthe number of named fungal cultures obtainablefor comparison.

To confirm that the isolate obtained from thesheathing mycorrhiza is the mycorrhizal fungus,synthesis of the mycorrhiza in aseptic conditions isdesirable. A technique for achieving this wasdeveloped by Melin (1921) and modified in avariety of ways by subsequent workers (Hacskaylo,1953; Bryan & Zak, 1961; Trappe, 1967b;Pachlewski, 1967; Mason, 1975).

This study is an attempt to characterize the

sheathing mycorrhizas of nursery-grown Piceasitchensis (Bong.) Carr. Levisohn (1965) lists thefollowing fungi as forming , true mycorrhizas'with nursery-grown P. sitchensis: Suillus booinus,Rhizopogon luteolus, Paxillus inoolutus, Sclero-derma vulgaris (= S. aurantium), Mycelium radicis-nigrostrigosum. In addition, 'pseudomycorrhizas'were formed by Mycelium radicis-atrovirens andRhizoctonia-like mycelia. The latter were des-cribed by Levisohn (1954) as being aberrant rootinfections characterized by the absence of a sheathand the presence of a very coarse intercellular net.Mikola (1965) regards these mycorrhizas as beingformed by strains of the same fungus that formsectendotrophic mycorrhizas with nursery-grownpine. Levisohn (1954) and Laiho (1965) havefound them to be common in many nurseries.

The presence of mycorrhizal associations in P.sitchensis is not always apparent from the externalappearance of the root system as it usually is inother conifers (Levisohn, 1965). Therefore micro-scopic examination is necessary to determineboth the presence and type of mycorrhizalinfection.

MA TERIALS AND METHODS

P. sitchensis seedlings (up to 1 year old) andtransplants (1 year old seedlings transferred totransplant beds for a further year) were obtainedat 2 month intervals from October 1975 toOctober 1977 from the Forestry CommissionExperimental Nursery at Bordon in Hampshire.These were removed from the nursery togetherwith surrounding soil and transported immediatelyto the laboratory. Lateral roots were then dissectedfrom the main root axis and representativemycorrhizal short roots were examined andcharacterized macroscopically. The short rootswere then fixed in formalin-acetic-alcohol. Micro-scopic characterization was based upon transversesections 6 /lm thick cut on a freezing microtome

0007-1536/79/2828-5120 $01.00 © 1979 The British Mycological Society

118 Mycorrht'zas of Picea sitchensis

and plan views of the sheath obtained from rootsquashes stained with lactofuchsin. Mycorrhizalroot material was prepared for photomicrographyby fixing in 4 % (vIv) glutaraldehyde solutionwith 0 '1 M sodium cacodylate buffer (pH 7'2).The material was then dehydrated and embeddedin Spurr's (1969) hard formulation resin. Sections(1 p,m) were cut with a glass knife and stained withMallory's stain (Richardson, Iarett & Finke,1960).

Prior to isolation, mycorrhizal short roots wereagitated in 0'003 % (v/v) Tween 80 on a benchflask shaker for 15 min to remove soil debris.The roots were then agitated for a further 15 minin distilled water, surface-sterilized by immersionin 30% (vI v ) hydrogen peroxide for 10 sec andwashed again by agitation for 15 min in each ofthree changes of sterile distilled water. Individualmycorrhizas were then plated out on modifiedMelin Norkrans medium (Marx, 1969, 1975)containing 15 p,g/ml aureomycin and incubated at20 °C.

Attempts were made to isolate from sporocarpsfound in the vicinity of seedlings and transplants,by plating out pieces of sporocarp tissue ontomodified Melin Norkrans medium and incubatingat 20 ° .

Aseptic synthesis of sheathing mycorrhizas wasattempted using the technique developed by Mason(1975) . Sterile glass vials containing a slope of17 ml ofIngestad's (1971) inorganic salts medium,containing phosphorous at a level of 6 ppm andamended with 10 g/l glucose, SO p,g/l thiaminehydrochloride and 10 gIl agar were inoculatedwith an agar plug cut from the edge of the testcolony. Next to the fungal inoculum was placedan aseptic P. sitchensis seedling. The vials werethen fitted with a plastic cap containing an airvent plugged with cotton wool and incubated at20° under Thorn C Gro-lux ' fluorescent tubesgiving a light intensity of 2200 Ix with a photo-period of 18 h. The seedlings were examined atregular intervals for mycorrhizal development.

Aseptic seedlings were grown from seedssterilized in 30 % (vIv) hydrogen peroxide for30 min (Trappe, 1961), plated out on 1 % (w/v)water agar and incubated at 20°.

RESULTS

Macroscopic and microscopic analysis of the rootsystems of nursery-grown P. sitchensis seedlingsand transplants revealed seven distinct types ofsheathing mycorrhizas. In all cases the overallroot form is racemose.

(1) P . sitchensis-s-Thelephora terrestris (Ehrh.)Fr. The individual mycorrhizas are grey-brown

and cylindrical. The sheath is up to 45 utt: wide,prosenchymatous and made up of septate inter-woven hyphae 2-7 l im diam with cells up to16 pm long. Single clamp connexions occurinfrequently on hyphae near the sheath surface.The epidermal and some of the outer corticalcells of the root are pigmented, distorted anecontain int racellular hyphae. The Hartig ntdextends to the endodermis and varies from 7 pmwide between the epidermal cells to 3 lim widebetween the cortical cells (Fig. 1).

Two types of hyphae radiate infrequently fromthe smooth sheath surface. The first type arefiliform, 2-3 p,m diam and up to 100 pm long.These hyphae are septate with clamp connexionsupon the distal septa. The second type are ofindeterminate length, 2-4 p,m diam with frequentsingle clamp connexions. Undifferentiatedrhizomorphs (Townsend, 1954) occur infrequently,up to 35 p,m diam and of indeterminate length.They consist of a central core of tortuous hyphae4-5 p,m wide with a clamp connexion at eachseptum. Radiating from the central core arehyphae similar to those found on the sheathsurface.

(2) P. sitchensis +Laccaria laccata (Seop. ex Fr.)Cooke. The individual myeorrhizas are lightbrown and cylindrical. The sheath is up to 25 p,mwide, prosenchymatous and made up of tightly-interwov en frequently forked septate hyphae,2-3 lim diam with cells up to 15 p,m long. Thehyphae appressed to the epidermal cells are lobateand up to 9 /tm wide. The epidermal cells arepigmented, distorted and contain intracellularhyphae. The Hartig net extends to the endodermisand varies from 4 p,m wide between the epidermalcells to 2 p.m wide between the cortical cells(Fig. 2).

Hyphae, 2-3 '5Itm diam, radiate infrequentlyfrom the smooth sheath surface. These hyphaeare tortuous with frequent elbow-like protrusionsand a single clamp at every septum.

(3) P. sitchensis+isolate Ba. The individualmycorrhizas are yellow-brown and cylindrical.The sheath is up to 35 pm wide, prosenchymatousand made up of tightly-interwoven septate sinuatehyphae, 1'5-3/tm diam with cells up to 30 limlong. The epidermal and outer cortical cells arepigmented and distorted. The Hartig net extendsto the endodermis and is 1-2 flm wide (F ig. 3).

Verrucose hyphae, 2'5/lm diam with a clampconnexion at every septum, radiate infrequentlyfrom the smooth sheath surface.

(4) P. sitchensis+ isolate B1• The indiv idualmycorrhiza s are yellow-brown and tomentosedue to copious loose hyphae and undifferentiatedrhizomorphs. The sheath is prosenchymatous and

G. W. Thomas and R. M. Jackson 119

Key to Figs. 1-7. A, Fungal sheath; B, Hartig net; C, Intracellular hyphae; D, hyphae surrounding mycor-rhiza; P, pigmented hyphae; H, hyaline hyphae.

Fig. 1. T. S. of P. sitchensisf T, terrestris (Type 1) mycorrhiza (x 582).

Fig. 2. T. S. of P. sitchensis/L. laccata (Type 2) rnycorrhiza (x 582).

120 Mycorrhizas of Picea sitchensis

"fT-- -A

Fig. 3. T. S. of P. sitchensis/isolate B, (Type 3) mycorrhiza (x 582).

,

3

Fig. 4. T. S. of P. sitchensis/isolate B1 (Type 4) mycorrhiza (x 582).

G. W. Thomas and R. M. Jackson 121

D---~"

"

c

I'

II~

Fig. 5. T. S. of P. sitchensis/isolate S, (T ype 5) mycorrhiza ( x 582).

"::::;r--"';-:'-- C

A

Fig. 6. T. S. of P. sitchensis/ isolate Az (T ype 6) mycorrhiza ( x 582).

6

122 Mycorrhizas of Picea sitchensis

Fig. 7. T. S. of P. sitchensis/isolate A3 (Type 7) mycorrhiza (x 582).

Fig. 8. ChIamydospores found associated with Type 6 mycorrhizas, (x 582) with verrucose walls.

G. W. Thomas and R. M. Jackson 123variable in width from 2 to 12 p,m, in some areasof the root surface consisting of a single layer ofhyphae. The hyphae are septate, frequentlyforked and lobed, 2-3'5 p,m diam with cells up to20 p,m long. The Hartig net extends to the endo-dermis and is 2-3 p,m wide with a beaded appear-ance. The epidermal and outer cortical cells of theroot are pigmented and distorted (Fig. 4).

Hyphae, 2-2'5 p,m diam, with yellow spinulosecell walls and frequent clamp connexions, radiateout from the sheath surface. The undifferentiatedrhizomorphs are of indeterminate length and up to60 p,m diam. They consist of a central core oftightly-interwoven hyphae, 4 p,m diam, withyellow cell walls and frequent clamps. Thiscentral core is surrounded by loosely-interwovenhyphae 3 p,m diam with yellow spinulose cell wallsand frequent single clamp connexions.

(5) P. sitchensis+isolate 84, The individualmycorrhizas are black-brown, clavate with manyblack-brown hyphae radiating from the sheathsurface. The sheath is up to 16 p,m wide, pro-senchymatous and frequently two layered. Bothlayers are made up of septate sinuate hyphae, 1-3 p,mdiam with cells up to 7 p,m long. The inner layeris hyaline, the outer dark brown, and when themycorrhiza is first formed the hyaline layer makesup the major part of the sheath. As it matures thewhole of the sheath becomes dark brown. TheHartig net in newly-formed mycorrhizas and in theproximal portion of the mature mycorrhiza is hya-line, penetrates to the endodermis and is 1-2 p,mwide. In the older portion of the mycorrhiza theHartig net between the epidermal cells and outercortical cells is pigmented (Fig. 5).

Radiating from the sheath surface are septateblack brown verruculose hyphae 2 p,m diam witha basal swelling of 3 p,m diam.

(6) P. sitchensis+isolate Az. The individualmycorrhizas are brown and cylindrical. Thesheath is up to 23 p,m wide, prosenchymatous andmade up of septate interwoven doliiform hyphae,3-5 p,m diam with cells up to 15 p,m long. Theepidermal cells and the outer two to four layers ofcortical cells are pigmented, distorted and containintracellular hyphae. The Hartig net extends to theendodennis and varies from 8 p,m wide betweenthe pigmented cells to 2 p,m between the corticalcells (Fig. 6).

Radiating from the smooth sheath surface are in-frequent smooth to verrucose thick-walled septatehyphae, 2-4 p,mdiam. Occasionally these hyphae areterminated by spherical chlamydospores (Fig. 8),which are thick-walled, yellow to dark brown,verrucose, 20-70 p,mdiam supported by two to fiveswollen cells.

(7) P. sitchensis+isolate As. The individualmycorrhizas are light brown and tortuous. Thesheath is up to 25 p,m wide, prosenchymatous andmade up of septate interwoven doliiform hyphae,4-5 p,m diam with cells up to 10 p,m long. Theepidermal and some of the outer cortical cells arepigmented, distorted and contain intracellularhyphae. The Hartig net extends to the endodermisand varies from 10 p,m wide between the pig-mented cells to 3 p,m between the cortical cells.Radiating from the smooth sheath surface areinfrequent smooth to verrucose thick-walledseptate hyphae, 2-4 p,m diam. Occasionally thesehyphae are terminated by chlamydospores similarto those described above.

Of the seven types of sheathing mycorrhizasdescribed above only two were found associatedwith sporocarps, Type 1 with those of T. terrestrisand Type 2 with those of L. laccata. Types 2, 3and 4 sheathing mycorrhizas were found associatedonly with transplants while Types 1, 5, 6 and 7were found with both transplanted and seedbedP. sitchensis, The majority of transplanted andseedling sheathing mycorrhizas were of Type 6 or7, many trees having only these mycorrhizas.When the other types of mycorrhizas were foundthey not only infected newly-formed short rootsbut also replaced Types 6 or 7 from older shortroots. The result of such replacement was shortroots with a distal portion of Type 6 or 7 and aproximal portion of the other types.

Types 2, 3 and 4 occurred very infrequently,usually being confined to a small portion of thetransplant bed, frequently localized upon a fewroots of an individual transplant. Type 1 occurredmore frequently but was not widespread, occurringrather as islands of infection in the transplant bedsand sporadically in the seedbeds. Type 5 occurredsporadically on both transplants and seedlings.

Isolates were obtained from all of the sheathingmycorrhizas and the sporocarps of T. terrestrisand L. laccata. Only those with hyphae resemblingthose of the original mycorrhizas were used toattempt aseptic synthesis.

The synthesized mycorrhizas did not initiallyclosely resemble the naturally-occurring mycor-rhizas. The fungal sheath became much wider,up to 80 p,m in the case of the isolate obtainedfrom Type 4. This enlarged sheath in all casescontained the outer two to three tiers of corticalcells which were widely separated by fungal tissueand frequently contained intracellular hyphae.No Hartig net was formed in any of the synthesizedmycorrhizas. However when glucose was omittedfrom the medium the mycorrhizas formed closelyresembled those found in the nursery.

We thank the Forestry Commission for thesupply of experimental material and the N.E.R.C.for a studentship held by one of the authors(G.W.T).

Mycorrhizas of Picea sitchensis

root infections. However, Mikola (1965) andLaiho (1965) have shown that fungi isolated fromsheathing mycorrhizas of nursery-grown sprucesimilar to Type 6 and 7, and termed 'E strains',gave rise to ectendomycorrhizas with nursery-grown pine and significantly improved growth.Wilcox, Ganmore-Neumann & Wang (1974)described an E-strain fungus isolated fromectendomycorrhizas of red pine which producedterminal chlamydospores in culture similar tothose found associated with Type 6 and 7 rnycor-rhizas and in cultures of Type 6 and 7 isolates.The fungi forming Type 6 and 7 mycorrhizasprobably belong to the E-strain group, which hasa widespread distribution. Chlamydospores attrib-uted to them are frequently found (Mosse &Bowen, 1968; Redhead, 1977; Hall, 1977) andmycorrhizas formed by them are dominant inmany nurseries (Laiho, 1965).

Mikola (1965) has shown with trials beyondthe nursery stage that the ectendomycorrhizas arereplaced almost immediately by more normalsheathing mycorrhizas. Replacement of thesheathing mycorrhiza form of these E-strain fungihas been shown, in our work, to occur in thenursery even in the first year of growth. Thisreplacement is sporadic, the OCcurrence ofbasidiomycete mycorrhizas among transplants isonly occasional, similar to that described byTrappe & Strand (1969). One of the first replace-ment basidiomycetes is T. terrestris; this fungus(Schramm, 1966; Marx et al., 1970) is one of themain initial sheathing mycorrhizal fungi found inwasteland and sterile soil.

The similarity of the microscopic features ofmycorrhizas formed by individual fungal speciessuch as T. terrestris and L. laccata with differenthost plants agrees with the findings of Zak (1973,1976). Microscopic features of mycorrhizas canthus be used as an effective method of classifica-tion. This is very important in the case of P.sitchensis since mycorrhizal infection is notobvious from macroscopic appearance and exten-sive sectioning of short roots and microscopicexamination is required to ascertain the presenceand type of mycorrhizal infection.

This study has shown that growth medium hasa pronounced effect upon synthesized mycor-rhizas, thus great care must be taken in relatingmycorrhizas synthesized aseptically with thoseoccurring in nature.

124

DISCUSSION

It was impossible to distinguish the type ofmycorrhiza from macroscopic features, exceptfor Types 4 and 5. Mycorrhizal classification wastherefore based upon the microscopic character-istics of the sheath, associated hyphae andrhizomorphs of each type.

The fungi of Type 1 and 2 were identified asT. terrestris and L. laccata respectively, based onthe circumstantial evidence of finding the mycor-rhizas in close proximity to sporocarps, andcomparisons of microscopic features of eachmycorrhiza with those described by other workersfor the same fungi with different host plantspecies. Type 1 mycorrhizas closely resemblemicroscopically those described by Fassi &Fontana (1966) for T. terrestrisfPinus strobus, andMarx & Davey (1969) for T. terrestrisf Pinusrigida. Isolates obtained from Type 1 mycor-rhizas closely resembled that obtained from asporocarp of T. terrestris found near the mycor-rhizal roots, and were very similar to the isolatesdescribed by Zak & Marx (1964) and attributed toT. terrestris.

Type 2 closely resembled L. laccata]P. strobusmycorrhizas described by Fassi & Fontana (1966).The isolates obtained from a sporocarp of L.laccata were very similar microscopically toisolates obtained from Type 2 mycorrhizas. Noneof the remaining fungi have been identified;Types 3 and 4 are formed by clamp-bearingbasidiomycetes, while Types 5, 6 and 7 are formedby septate but non-clamp-bearing fungi.

Type 5 mycorrhizas occur infrequently andsuperficially resemble those described by Hatch(1934) and attributed to MyceZium radicis-atrovirens Melin. Hatch considered this fungus tobe a dark mycelium parasitizing a hyaline rnycor-rhiza. Richard, Fortin & Fortin (1971) noted themicroscopic characteristics of M. radicis-atrovirensinfection of aseptically grown seedlings of Piceamariana as intracellular penetration of corticalcells of the roots; microsclcrotia formation withincortical cells; deeply-penetrating hyphae oftenhyaline and bulbous. This microscopic descriptiondoes not fit that of Type 5 mycorrhizas whichhave a well-developed sheath and Hartig net, andintracellular penetration limited to the pig-mented distorted epidermal cells. Synthesis usingpure cultures obtained from Type 5 mycorrhizasgave rise to mycorrhizas that varied from havinghyaline to totally-pigmented sheaths. Thus Type 5can be considered to be a true sheathing mycor-rhiza.

Mycorrhizas similar to Types 6 and 7 weredescribed by Levisohn (1954) as being aberrant

G. W. Thomas and R. M.Jackson

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(Accepted for publication 10 November 1978)