Effect of Laccaria bicolor strains inoculated on Douglas-fir ( Pseudotsuga menziesii ) several years after nursery inoculation

Effect of Laccaria bicolor strains inoculated onDouglas-fir (Pseudotsuga menziesii) several yearsafter nursery inoculation

M.-A. Selosse, D. Bouchard, F. Martin, and F. Le Tacon

Abstract: In the Saint-Brisson experiment conducted in central France, the American strain of the ectomycorrhizal fun-gus Laccaria bicolor (Maire) P.D. Orton S238N and the French strainL. bicolor 81306 inoculated on containerizedDouglas-fir (Pseudotsuga menziesii(Mirb.) Franco) seedlings increased by 60% the total volume of wood produced 8years after outplanting as compared with uninoculated but naturally mycorrhizal trees. The two strains introduced 10years before in the inoculated plots are still present and dominant; they did not prevent the colonization of Douglas-firroots by naturally occurring ectomycorrhizal fungi but allowed for the establishment of a very diversified symbioticmicroflora. Eight to 12 years after outplanting, all the Douglas-fir plots were colonized byLaccaria laccata(Scop.:Fr.)Cooke orL. bicolor strains, as well as some other species, independently of the nursery treatments. With one exceptionin one plot, the presence of indigenous genets in the control treatments may have prevented the vegetative colonizationof the inside of the noninoculated plots by the two introduced strains.

Résumé: Dans l’essai de Saint-Brisson installé dans le centre de la France, la souche américaine du champignon ecto-mycorhizienLaccaria bicolor (Maire) P.D. Orton S238N et la souche françaiseL. bicolor 81306 inoculées à des semisde Douglas (Pseudotsuga menziesii(Mirb.) Franco) en conteneurs ont augmenté de 60% le volume de bois produit 8ans après la transplantation en comparaison avec des semis non inoculés et naturellement mycorhizés. Les deux sou-ches inoculées sont toujours présentes et dominantes dans les placeaux où elles ont été introduites 10 ans auparavant.Cependant, 8–12 ans après la transplantation, tous les placeaux de Douglas sont colonisés par des souches deL. bico-lor ou deLaccaria laccata(Scop.:Fr.) Cooke, quel que soit le traitement. La présence des souches introduites dans lesplaceaux inoculés n’empêche pas la colonisation des racines de Douglas par des champignons ectomycorhiziens natu-rels et permet l’établissement d’une microflore symbiotique diversifiée. Bien qu’il y ait une exception, la présence deces souches naturelles dans les témoins pourrait expliquer l’absence d’invasion du centre de ces placeaux par les deuxsouches introduites. Selosse et al. 371

Introduction

There are now many examples of the ability of inoculatedectomycorrhizal fungi to improve growth of trees in nurser-ies and after field outplanting. Experimental studies, mostlyin temperate forest nurseries in Europe and in North Amer-ica, have shown that fungal pure culture or spore inoculationcan favour development of mycorrhizae of effective fungalspecies (Le Tacon and Bouchard 1986). Knowledge of theability of specific ectomycorrhizal fungi to improve the sur-vival and early growth of various plantation species in thefield have been gained over the past decades.

Large increases in growth of pines have been recorded inthe field, examples being the 20–40% increases in heightgrowth ofPinus radiataD. Don inoculated withRhizopogonand Suillus species (Theodorou and Bowen 1970) and the25–100% increases in growth of various pine species inocu-lated withPisolithus tinctorius(Mich.:Pers.) Coker & Couch

and established on several routine reforestation sites inFlorida and North Carolina (Marx et al. 1977). Although in-oculation withPisolithus tinctoriushas also been shown toimprove the growth ofPinus caribaeaMorelet at forest sitesin Liberia (Marx et al. 1985) and the Congo (Delwaulle etal. 1987), it has been ineffective in increasing growth inother major forest areas in cooler regions (Castellano 1988;Perry et al. 1987).Laccaria bicolor (Maire) P.D. Orton andLaccaria laccata(Scop.:Fr.) Cooke, ruderal and pioneer spe-cies (Last et al. 1987) that colonize nurseries (Henrion et al.1994) but also persist in ageing natural forest stands (Baar etal. 1994), have been proven suitable for tree inoculation inNorth America (Sinclair et al. 1982; Kropp and Langlois1990; Buschena et al. 1992).

In France, Douglas-fir (Pseudotsuga menziesii(Mirb.)Franco) seedlings mycorrhizal with an AmericanL. bicolorstrain showed contrasting responses after outplanting on pre-viously forested sites or on lands with a long history of cul-tivation. Different effects of inoculation on total height wereobserved among two previously cultivated sites having simi-lar soils but differing in dominant plant species (Le Tacon etal. 1992). On a grassland site,L. bicolor that had success-fully infected seedling roots in the nursery disappearedshortly after outplanting (Longechaud experiment, Le Taconet al. 1992). On another site invaded by bracken (Pteridiumaquilinum), 3 years after planting, seedlings inoculated

Can. J. For. Res.30: 360–371 (2000) © 2000 NRC Canada

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Received June 21, 1999. Accepted October 24, 1999.

M.-A. Selosse, D. Bouchard, F. Martin, and F. Le Tacon.1

Équipe de microbiologie forestière, Institut national de larecherche agronomique, Centre de Nancy, 54 280Champenoux, France.

1Corresponding author. e-mail: [email protected]

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with L. bicolor S238N or L. bicolor 81306 were signifi-cantly taller than seedlings naturally mycorrhizal in the nurs-ery with Thelephora terrestrisPers. (Ballandeix experiment;Le Tacon et al. 1992). Furthermore, at that time,L. bicolorfruiting bodies were 10-fold more frequent under inoculatedtrees than under control trees (Le Tacon et al. 1988),strongly suggesting the persistence of inoculated strains.Similarly, in northern Spain, Alvarez et al. (1996), haveshown that L. bicolor was able to improve Douglas-firgrowth after outplanting. The field responses appeared to beinfluenced by the site characteristics, the vigour of the nativeectomycorrhizal fungi and the quality of the naturalinoculum in the nursery.

In contrast to previously cultivated sites, forest sites gen-erally have a high level of naturally occurring ectomycor-rhizal fungi that can colonize the roots systems (Guinberteauet al. 1989). By competing with introduced fungi or rapidlyinfecting uninoculated seedlings, these fungi may be ex-pected to reduce the likelihood of responses to nursery inoc-ulation. For example, in a study of outplanted Douglas-fir byBledsoe et al. (1982), the inoculatedL. laccata and Hebe-loma crustuliniforme(Bull. ex St. Amans) Quèl. did not per-sist and newly formed roots were colonized by indigenousfungi. However, Buschena et al. (1992) found that coloniza-tion of black spruce (Picea mariana (Mill.) BSP) byL. bicolor persisted after 2.5 years in the field. Studies in thewestern United States (Castellano 1988) and in France(Villeneuve et al. 1991; Le Tacon et al. 1992) have shownthat inoculation of Douglas-fir seedlings with selected fun-gal isolates significantly increased their survival and (or)early growth on recently clearcut forest sites compared withnaturally inoculated seedlings. In a detailed study, Ville-neuve et al. (1991) examined the persistence, spread, and ef-fectiveness of an introducedL. bicolor isolate and ofnaturally occurring mycorrhizal fungi on Douglas-fir for 2years after outplanting on a clearcut forest site. The intro-duced strain increased height growth compared withuninoculated plant, persisted on old roots and, together withindigenousLaccaria species, was able to colonize a majorproportion of newly formed roots. In this study,Thelephoraand Rhizopogonthat had infected roots of uninoculatedseedlings in the nursery did not persist in the field, andnewly formed roots became infected with indigenous fungithat included species ofLaccaria, Cenococcum, Paxillus,Scleroderma, and Hebeloma. Positive growth responses toinoculation and persistence of introduced fungi on forestsites in these studies occurred despite the presence of this di-verse ectomycorrhizal fungal population.

Nevertheless, surveys based on morphotyping have to beconsidered with care, since morphological identification ofectomycorrhizae may be misleading (Mehmann et al. 1995).In addition, morphological identification methods cannotdistinguish fungal inoculants at the strain level.

Usefulness of analysis based on polymerase chain reac-tion to identify fungal strain on single root tips or on sporo-phores have been demonstrated (Gardes et al. 1991; Henrionet al. 1994; Di Battista et al. 1996; Gryta et al. 1997 and ran-dom amplified polymorphic DNA (RAPD) can even distin-guish genets (Selosse et al. 1998a). DNA typing haspreviously been used for demonstrating the persistence oftwo L. bicolor strains in a Douglas-fir plantation set up 10

years ago in the centre of France on a clearcut forest stand(Selosse et al. 1998a and 1998b). Here we report on the ef-fect of L. bicolor inoculation on Douglas-fir growth andwood production in this experiment. To explain these data,we further analysed the overall fungal colonization patternas well as the belowground community of the plantation.

Materials and methods

Fungal strainsTwo strains of L. bicolor were used in this study. The

strainL. bicolor S238N was isolated by J. Trappe and R. Molina in1976 from a basidiome underTsuga mertensiana(Bong.) Carr.at Crater Lake National Park, Oregon, U.S.A. This isolate wasformerly accessioned and distributed asL. laccata. According toribosomal DNA restriction patterns and culture morphology(Armstrong et al. 1989), it was reclassified asL. bicolor. A subcul-ture was transferred to the Institut national de la rechercheagronomique fungal collection (Nancy, France) in March 1980 andthen subcultured every 2–3 months on solid modified Pachlewski’smedium (7.3 mM KH2PO4, 2.7 mM diammonium tartrate, 7.3 mMMgSO4·7H2O, 100 mM glucose, 2.9 mM thiamine-HCl, and 1 mLof a trace element stock solution (Kanieltra Co.) in 2% agar) inPetri dishes at 25°C. The strainL. bicolor 81306 was isolated byDenise Lamoure in 1981 from a basidiomeunder Douglas-fir artifi-cially introduced at Barbaroux (Haute-Vienne, France). This isolatewas then subcultured every 2–3 months on solid modifiedPachlewski’s medium in Petri dishes at 25°C.

Production of ectomycorrhizal seedlingsContainerized Douglas-fir seedlings from an American prove-

nance (Ashford) were grown for 2 years in the French nursery ofPeyrat-le-Chateau (seedlings of the Mieville nursery, also studiedby Selosse et al. (1998a), were not considered in the presentstudy). In the 1985 spring, 1-L containers were filled with thesandy loam soil of the nursery, which was a brown podzolic soildeveloped on granite, chemically improved after 10 years of inten-sive fertilization (Table 1). Before filling the containers, the soilwas fumigated or not with methyl bromide under clear polyethylene

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Nursery Plantation

Particle size analysis (‰)Clay n 279Fine silt n 206Coarse silt n 77Fine sand n 83Coarse sand n 355Total C (‰) 43 45Organic matter (‰) 74.0 77.4Total nitrogen (‰) 3.3 2.32C/N 13.01 19.39pH in water (1:5 soil to H2O) 5.6 4.2P2O5 (‰)a 0.50 0.21Exchangeable calcium (cmol (+)/kg) 4.2 0.1Exchangeable magnesium (cmol (+)/kg) 1.03 0.08Exchangeable potassium (cmol (+)/kg) 0.45 0.195Exchangeable aluminium (cmol (+)/kg) n 6.8Total copper (ppm) n 19.1

Note: n, not measured.aExtracted with NaOH 10–1 M and H2SO4 5 × 10–2 M.

Table 1. Chemical analyses of the nursery soil and of the A1 ho-rizon of the plantation soil.



(100 g/m2). The containers with fumigated soil were inoculated ornot with ectomycorrhizal strains grown in a vermiculite–peat mois-ture nutrient mixture for 2 months at 25°C, using techniques de-scribed by Marx and Bryan (1975). In each inoculated container,100 mL of nonleached inoculum were mixed with the fumigatedsoil. Four treatments were applied as follows without any fertiliza-tion: (i) nonfumigated, noninoculated soil; (ii ) fumigated, non-inoculated soil; (iii ) fumigated soil and inoculation withL. bicolorS238N; and (iv) fumigated soil and inoculation withL. bicolor81306. Note that the first two treatments allow colonization by nat-urally occurring ectomycorrhizal species during nursery growth.The experiment was a randomized complete design with four repli-cations for each treatment and sixty 1-L containers per plot. Fiveseeds were placed in each container. One month after germination,Douglas-fir seedlings were thinned to one per container. Then theywere maintained 2 years in the nursery. At the end of the nurseryphase, the height of all seedlings was measured, and 10 containersper plot were randomly selected for mycorrhizal assessment.

Mycorrhizal assessment at the end of the nursery phaseAfter 2 years growing in the nursery, the mycorrhizal status of

10 seedlings per plot was assessed. After lifting, the roots of eachseedling were separated from soil, washed, and cut into pieces1 cm long. Pieces of roots were randomly picked and examined forectomycorrhizal development under a dissecting microscope. Allshort roots up to 200 were counted in this subsample and thedifferent morphotypes were counted separately (Laccaria,Thelephora, Rhizopogon, and others).

Field experiment site and designIn March 1987, containerized seedlings were planted without

any fertilization on a recently clearcut site in Saint Brisson. Thisstand is located in the Nièvre (centre of France), at an elevation of630 m and was previously a mixed forest of beech, oak, and birchon a brown podzolic soil over granite. The A1 horizon is acid witha low level of exchangeable cations (Table 1). Average annual pre-cipitation is 1100 mm/year; mean annual temperature is 9.5°C. Thestand was divided into plots of 7.2 m × 7.2 meach separated by a3-m unplanted buffer zone. Forty-nine containerized seedlingswere hand planted per plot. As in the nursery, the experiment was arandomized complete design with the same four treatments andwith four replications. The ground was hand cleared during thesummers of 1987, 1988, and 1990. The weeds, shrubs, and unde-sired tree species were removed to assure the good establishmentand the early growth of Douglas-fir seedlings.

The plantation was randomly thinned twice. In February 1992,trees of one raw on two were cut and, in February 1995, onetree on two was removed. Sixteen trees per plot remained in 1995.Total and annual shoot lengths as well as collar or stem diametersof all trees were measured at the end of almost each growingseason. The volume of each thinned tree was estimated by multi-plying its height by the square of the diameter measured at halfof the height. For the remaining trees an equation relating thestem diameter to the volume was calculated for each of the fourtreatments.

Mineral elements concentration in the needlesDuring winter 1987–1988, analysis of needle nutrient concentra-

tions was performed for N, P, K, and Cu. Total N and P in the tis-sues were extracted using concentrated H2SO4, 30% H2O2, andmetallic selenium as a catalyst and measured by an automaticcolorimeter (Technicon model No. 321-74A). Total K and Cu wereextracted using concentrated perchloric acid and measured byemission spectrophotometry (Jobin and Yvon spectrophotometer).

Mycorrhizal assessment in the plantationIn May 1997, the mycorrhizal status of the trees was assessed

on one of theL. bicolor S238N inoculated plot by randomly taking17 cores of the upper 10 cm of soil with a cylinder of 0.220 dm3

(5 cm high by 7.5 cm diameter). The roots of each cylinder wereseparated from soil, washed, and cut into pieces 1 cm long. All themycorrhizae were examined for ectomycorrhizal development un-der a dissecting microscope and frozen for molecular typing. In ad-dition to Laccaria-like morphotype (single mycorrhiza, oftentortuous, 2–10 mm long, 1–2 mm wide, cottony textured, whitishto brown mantle, abundant emanating hyphae with abundant clampconnections), we distinguished nine other morphotypes: (i) Russula-or Lactarius-like type: single to pinnately branched, smooth, lightbrown mycorrhizae; (ii ) Russula- or Lactarius-like type: single topinnately branched, smooth, olive-coloured mycorrhizae, exclu-sively colonizing decaying wood; (iii ) unidentified single mycor-rhizae: cottony light brown mantle, no strands or rhizomorphs;(iv) unidentified single mycorrhizae: cottony white mantle, nostrands or rhizomorphs; (v) Amanita- or Cortinarius-like type: cot-tony white mantle, rhizomorphs with interconnected filaments;(vi) Piloderma croceumErikss. & Hjortst.-like type: cottonyyellow mantle, yellow strands; (vii) Chalciporus-like type:monopodial-pyramidal mycorrhizae with tortuous axis, cottonouspink reddish mantle, white strands; (viii ) Rhizopogon-like type:single to pinnately branched or tuberculate mycorrhizae, white tolight brown, rough mantle, abundant emanating hyphae withoutclamp connections, abundant strands forming mats; (ix) Cenococcum-like type: single mycorrhizae, rough and black, 1–3 mm long,black abundant emanating hyphae without clamp connections.

Sporophore assessmentDuring autumn 1987, i.e., one growing season after outplanting,

all Laccariasporophores were counted in all the plots, without mo-lecular analysis. From autumn 1995 to summer 1997, a survey ofthe various sporophores of ectomycorrhizal species was performed,in the centre of the experiment including buffer zones, on a subsetof 11 plots where all the treatments were represented (Table 2);Lac-caria sporophores were sparingly collected (one sporophore perpatch at least) or extensively (for a plot of each inoculated treat-ment, see Table 2, plot Nos. 8 and 10). They were mapped with aminimal precision of 10 cm and kept at –80°C before DNA typing.

DNA typingTotal DNA from frozen sporophores and mycorrhizae was ex-

tracted according to Henrion et al. (1994). The 25S–5S spacer(IGS1) and 5S–17S spacer (IGS2) of the ribosomal DNA were am-plified as described in Selosse et al. (1996). The mycorrhizal DNAwas analysed to amplify two stretches of the ribosomal DNA,namely the IGS1 and the total internal transcribed spacer (ITS, in-cluding the 5.8S sequence, using the primers ITS1F and ITS4B;Gardes and Bruns 1993). RAPD profiles were obtained from thesporophore DNA using the primers 174 (5′-AACGGGCAGC-3′)and 152-C (5′-CGCACCGCAC-3′) in the conditions described bySelosse et al. (1998a). Some of those sporophores were also ana-lysed in other studies using additional RAPD primers (Selosse etal. 1998a, 1999) and mitochondrial markers (Selosse et al. 1998b),but this did not challenge the genet counting reported here. When asporophore had the same pattern as an inoculant strain, additionalmarkers were used to ensure its identity (for markers of the strainS238N, see Selosse et al. (1998a); for markers of the strain 81306,see Selosse et al. (1999)). The mycorrhizal DNA was analysed byamplification of the IGS1 to assess the persistence of the inocu-lated strains on Douglas-fir roots. Amplification products andRAPD profiles were separated on 8% acrylamide gel and stainedusing ethidium bromide.

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Statistical analysisData were subjected to analysis of variance, and Duncan’s test

was used for the means comparison.

Results

Seedling growth and mycorrhizal status in nurseryAt the end of the two growing seasons in the nursery,

shoot height of seedlings inoculated withL. bicolor S238Nand L. bicolor 81306 did not differ but was significantlygreater than the one of control seedlings produced on fumi-gated or nonfumigated soil (Table 3). Seedlings growing onnonfumigated soil had a significantly lower shoot heightthan seedlings produced on fumigated soil (Table 3).

The mycorhizal status at the end of the nursery phasestrongly depended on the treatment. Control seedlings, pro-duced without fungal inoculation on nonfumigated soil, werecolonized byThelephora terrestris(74% of the root tips),Laccaria-like mycorrhizae (3.5%) andRhizopogonsp. (1.5%of the root tips). Control seedlings, produced without fungalinoculation on fumigated soil, were colonized byThelephoraterrestris (23% of the root tips) andRhizopogonsp. (1% ofthe root tips). Seedlings inoculated withL. bicolor S238Nshowed mainlyLaccaria-like mycorrhizae (82% of the roottips) with some someRhizopogon-like mycorrhizae (less that1% of the root tips). Seedlings inoculated withL. bicolor81 306 were mainly colonized byLaccaria-like mycorrhizae

(77% of the root tips) with someRhizopogon-like mycor-rhizae (less that 1% of the root tips).

Field performance after outplantingThe ectomycorrhizal status of the seedlings at planting did

not significantly affect survival (data not shown) but consid-erably modified the growth after outplanting. Inoculationwith the twoLaccariastrains resulted in a further growth in-crease (Figs. 1A and 1B) compared with noninoculatedtrees. The difference in height between the control seedlingsfrom fumigated and nonfumigated soil was no longer signif-icant by 1989. The twoLaccaria strains behaved similarlyand never statistically differed in terms of improvingDouglas-fir growth. Eight years after outplanting, the meanheight of the inoculated trees was 16% greater than that ofthe controls (Table 3). Initially, inoculated Douglas-fir plantshad an annual height growth higher than the noninoculatedones, but annual height growth tended to be the same in1992 and 1994 (Figs. 1C and 1D). Eight years afteroutplanting, volume yield per plot was over 60% greater forinoculated trees than for noninoculated ones (Fig. 2).

Mineral elements concentrationOne year after outplanting, despite their growth increase,

trees inoculated with the twoL. bicolor strains had the sameN, P, K and Cu concentration in their needles as controltrees (Table 4), suggesting that the growth increase induced

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On the plot and surrounding buffer zones

Nursery treatmentsPlotNo.

No. ofsporophores

Number of genets(indigenous + inoculated)

No. of genetsinside the plot

Naturally mycorrhizal seedlingswithout soil fumigation

1 77 16 + 81306 9

2 43 6 + S238N 43 18 7 + S238N 54 36 12 + S238N 3

Naturally mycorrhizal seedlingsafter soil fumigation

5 65 13 + 81306 6

6 103 8 + 81306 and S238N 81306 + S238N7 24 7 + S238N 4

Seedlings inoculated with 8* 269 8 + S238N 1 + S238NL. bicolor S238N 9 10 4 + S238N 2 + S238NSeedlings inoculated with 10* 380 17 + 81306 4 + 81306L. bicolor 81306 11 54 8 + 81306 4 + 81306Total for the whole analysed surface 880 80 + 81306 and S238N —

*An extensive sporophore collection has been performed (see Materials and methods), explaining the high sporophores numberscollected in these plots.

Table 2. Summary of the molecular analysis performed on 11 plots from the Saint Brisson plantation.

Nursery treatmentsNursery(1987)

Plantation(1994)

Naturally mycorrhizal seedlings without soil fumigation 22.7a 515aNaturally mycorrhizal seedlings after soil fumigation 28.3b 528aSeedlings inoculated withL. bicolor S238N 35.1c 595bSeedlings inoculated withL. bicolor 81306 36.7c 598b

Note: Means followed by different letters are significantly different at the 0.05 level.

Table 3. Mean height (cm) of the containerized seedlings at the end of the nursery phasein 1987, and in the plantation in 1994.



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Fig. 1. Growth of Douglas-fir containerized seedlings from the Peyrat-le-Château nursery before and after outplanting, depending on the mycorrhizal treatment. (A) Total treeheight; (B) tree height expressed as difference from the control; (C) growth of the shoot of the year; (D) growth of the shoot of the year expressed as difference from the con-trol. For each panel, treatments are as follows: (s) uninoculated, naturally mycorrhizal seedlings without soil fumigation; (d) uninoculated, naturally mycorrhizal seedlings af-ter soil fumigation; (u) seedlings inoculated withL. bicolor S 238 N; (n) seedlings inoculated withL. bicolor 81306.

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by the two inoculated strains did not entail any nutritionaldeficiency from these four elements.

Survey of ectomycorrhizal sporophoresOne year after outplanting,L. bicolor sporophores were

found only in inoculated plots (Table 5, Fig. 3A): 61% of thetrees previously inoculated withL. bicolor S238N producedfruit bodies against 44% of the trees inoculated withL. bicolor81306. MoreLaccaria sporophores were recorded in plotsinoculated byL. bicolor S238N than in plots inoculated

by L. bicolor 81306. NoL. laccatasporophores were foundin the inoculated plots nor in the noninoculated ones. Atoutplanting time,Laccaria fructification seemed to entirelydepend on artificial mycorrhizal inoculation from the nurs-ery. Monitoring of the aboveground fruiting bodies between1994 and 1997 showed a completely different situa-tion. L. laccataandL. bicolor sporophores were found everyyear in all plots, previously inoculated or not. It also re-vealed the presence ofChalciporus piperatus(Bull. ex Fr.)Bat. (relatively abundant and found in all the plots) and

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Fig. 2. Wood volumes per plot (dm3) of the four treatments in 1994, eight years after outplanting, including volume recovered afterthe two thinnings (1992 and 1994).

Nursery treatments N (%) P (%) K (%) Cu (ppm)

Naturally mycorrhizal seedlings without soil fumigation 2.17 0.241 0.60 4.75Naturally mycorrhizal seedlings after soil fumigation 2.10 0.196 0.59 4.25Seedlings inoculated withL. bicolor S238N 2.13 0.206 0.64 4.25Seedlings inoculated withL. bicolor 81306 2.15 0.196 0.66 5.00

Note: Means are not significantly different at the 0.05 level.

Table 4. Nutrient mineral concentration in winter 1987–1988.

Average no. per plot

Nursery treatmentsLaccaria spp.sporophores

Trees withLaccaria spp.sporophores

Naturally mycorrhizal seedlings without soil fumigation 0 0Naturally mycorrhizal seedlings after soil fumigation 0 0Seedlings inoculated withL. bicolor S 238 N 141 30 (61%)Seedlings inoculated withL. bicolor 81306 100 28 (44%)

Table 5. Sporophores survey performed in autumn 1987.



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Paxillus involutus(Batsch) Fr.,Amanita muscaria(L. exFr.) Hooker,Cortinarius sp., andInocybesp. (the last fourspecies being rare and only found in some plots).

DNA typing of the Laccaria spp. sporophoresA total of 880 Laccaria spp. sporophores was collected

from 1994 to 1997 on 11 plots of the experiment, includingbuffer zones (Table 2). Sporophores having identical RAPDand IGS patterns were considered as belonging to the samegenet (= genetic individual). This typing showed that thetwo inoculated strains persisted in all the plots where theyhave been previously introduced, fruiting all over the plotsand in adjacent buffer zones (Selosse et al. 1998a, 1999;Fig. 3B). We never foundL. bicolor S238N in plots inocu-lated with L. bicolor 81306 and vice versa. In all plots, in-digenous L. bicolor and L. laccata genets were found(Fig. 3B); on inoculated plots, they appeared more fre-quently in the buffer zones than inside the plots. Their num-bers were not significantly different on inoculated versusnoninoculated plots (Mann–WhitneyU test, P < 0.05).Sporophores of inoculated strains colonized the surroundingbuffer zones of noninoculated plots (Table 2) only whenadjacent to inoculated plots (data not shown). The two inoc-ulated L. bicolor strains were absent from all the non-inoculated plots but one, planted with seedlings fromfumigated nursery soil (Table 2, plot No. 6, and Fig. 3B;Selosse et al. 1999). In contrast to the inoculated strains, thesporophores of the indigenous genets formed small patches,with maximal distance between sporophores ranging up to3.3 m (Fig. 3B). Most genets appeared yearly or in morethan 1 year, forming up to 32 sporophores per year. Someothers were found in a single year, which may be due to se-lective sporophore sampling. Patches of indigenousLaccariagenets were infrequently overlapping but never split (withthe exception of genet G described in Selosse et al. (1998a)that was fragmented in three parts).

Mycorrhizal assessment of Douglas-fir inoculatedwith L. bicolor S238N

Ten ectomycorrhizal morphotypes were identified among1837 mycorhizal tips collected on aL. bicolor S238N inocu-lated plot (Table 6). Strikingly, no morphotype correspond-ing to the nursery-abundantThelephora-like type was found.The dominant morphotype was theLaccaria-like one(68.7% from 0 to 5 cm and 81.8% from 5 to 10 cm). Thismorphotype was identified under patches ofLaccaria sporo-phores but also in places where they never appeared.Chalciporus-like mycorrhizae were also found independ-ently of the presence ofC. piperatussporophores above-ground (Table 6), suggesting a loose relationship betweenabove- and below-ground populations. We were able to am-plify only 35 fungal IGS1 of the collected mycorrhizae (outof 370 molecularly analysedLaccaria-type mycorrhizae).

Three of them showed the IGS1 pattern ofL. bicolor S238N(data not shown) with its characteristic heteroduplexes(Selosse et al. 1996), further proving that the strainL. bicolorS238N was still colonizing the root systems of the inocu-lated trees.

Discussion

The experimental plantation of Saint-Brisson (Nièvre,France) showed that, several years after outplanting,Douglas-fir growth was significantly increased byL. bicolornursery inoculation (Fig. 1). This beneficial effect, lastingseveral years after outplanting extended the growth improve-ment observed in nursery (Table 3) and seems to be due tothe persistence of the two introduced strains. In another ex-periment where the introduced strain (L. bicolor S238N) hasnot survived, the beneficial effect observed during the nurs-ery phase completely disappeared after field transplantation(Le Tacon et al. 1992). The American strain (L. bicolorS238N) and the French strain (L. bicolor 81306) used fornursery inoculation were equally effective. Their inoculationincreased by 60% the total volume of wood produced 8years after outplanting (Fig. 2), as compared with uninocu-lated but naturally mycorrhizal trees from the plantation.The effects of ectomycorrhizal inoculation probably wouldhave been higher with larger buffer zones. Since completecolonization of the buffer zones by roots was achieved after5 or 6 years, border trees of the control plots flanking the in-oculated ones may have been colonized by the introducedstrains. The presence of sporophores of the inoculant strainsin buffer zones (Table 2, Fig. 3) suggests that they could col-onize the roots and improve the growth of some controltrees. Moreover, because of the increased growth of theinoculated Douglas-fir trees, we can speculate that the com-petition between trees for water use was greater in the inoc-ulated plots than in the controls. Similar results werereported by Duddrige et al. (1980), Boyd et al. (1986), andMarx et al. (1988) on loblolly pine (Pinus taedaL.) associ-ated withPisolithus tinctorius. However, Marx et al. (1988)suggested that root systems with abundantPisolithustinctorius ectomycorrhizae may be more capable of absorb-ing water from soil than roots associated with lessPisolithusor with other fungi. The lack of difference with control forthe shoot of the year after 1992 (Figs. 1C and 1D) supportsthese hypotheses of a higher water stress in inoculated plotsand possible colonization of control plots. To avoid the ef-fects of such water (if not nutrient) deficit in tree standsinoculated with efficient ectomycorrhizal strains, low planta-tion density, vigorous thinning, and a lower length of the ro-tation should be recommended.

The growth increase observed several years afteroutplanting in the inoculated plots suggests that the two in-oculated L. bicolor strains have survived and colonizednewly formed roots. The sporophores survey carried out 1

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Fig. 3. Map of the sporophores collected on four contiguous plots of the Saint Brisson plantation. (A) Sporophores collected in autumn1987 (see also Table 4); each circle represents a sporophore. (B) Sporophores collected in 1994–1997, with indication of the genetthey belong to: open circle,L. bicolor 81306 sporophores; solid circle,L. bicolor S238N sporophores; Greek or Roman letters, indige-nous genets. Plots are represented by squares (control seedlings, naturally mycorrhizal, either from fumigated soil (top left) or not (bot-tom right)), square with shaded border (seedlings artificially inoculated by the strain S238N) or square with broken line (seedlingsartificially inoculated by the strain 81306).



year after outplanting (Table 5), showing sporophores onlyunder inoculated trees, suggests a good mycorrhizal infec-tion by the two strains and their survival, even without usingmolecular tools. A strikingly similar pattern of fructificationwas observed 1 year after outplanting under inoculated andnoninoculated trees planted in a previously cultivated soil

(Ballandeix experiment; Le Tacon et al. 1992): 60% of theDouglas-fir inoculated withL. bicolor S238N producedsporophores, as did 42% of those inoculated withL. bicolor81306, whereas no fructification was found in the controls(compare with Table 5). Abundant fruiting of inoculated ge-nets has also been demonstrated in nursery (Henrion et al.

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368 Can. J. For. Res. Vol. 30, 2000

Core No.

Mycorrhizal short roots (no./dm3) Laccaria-like mycorrhizae(%, whole core)Laccaria-like Other typesa

1b Upper part 4.5 None 100Lower part 0 None

2b Upper part 0 None —Lower part 0 None

3b Upper part 762.5 C: 13.6 98.6Lower part 796.9 D: 40.7

4b Upper part 13.6 H: 49.8 81.8Lower part 230.9 H: 4.5

5c Upper part 502.6 G: 787.8 38.0Lower part 36.2 G: 90.6

6c Upper part 27.2 None 100Lower part 27.2 None

7c Upper part 126.8 None 100Lower part 158.5 None

8c Upper part 31.7 None 100Lower part 0 None

9b Upper part 928.9 G: 36.2 97.1Lower part 285.2 None

10c Upper part 72.4 B: 45.3 42.8Lower part 9.1 H: 63.4

11b Upper part 13.6 G: 13.6; B 9.1 27.3Lower part 0 B: 13.6

12b Upper part 36.2 G: 40.7; I: 90.6 21.6Lower part 0 None

13b,d Upper part 190.2 I: 31.7; F: 262.6; A: 194.7 39.0Lower part 122.2 None

14b,d Upper part 824.1 None 100Lower part 0 None

15b Upper part 421.1 None 98.5Lower part 172.1 H: 9.1

16b Upper part 122.2 H: 77 70.9Lower part 153.9 E: 36.2

17b Upper part 27.2 None 36.6Lower part 90.6 E: 203.7

No. of assessedmycorrhizal short roots

1367 470

Mean percentage ofLaccaria-like mycorrhizae

74.4

aA, Russula- or Lactarius-like type: single to pinnately branched, smooth, light brown mycorrhizae; B,Russula- or Lactarius-liketype: single to pinnately branched, smooth, olive-coloured mycorrhizae, exclusively colonizing decaying wood; C, unidentified singlemycorrhizae: cottony light brown mantle, no strands or rhizomorphs; D, unidentified single mycorrhizae: cottony white mantle, nostrands or rhizomorphs; E,Amanita- or Cortinarius-like type: cottony white mantle, rhizomorphs with interconnected filaments; F,Piloderma croceum-like type: cottony yellow mantle, yellow strands; G,Chalciporus-like type: monopodial-pyramidal mycorrhizaewith tortuous axis, cottony pink reddish mantle, white strands; H,Rhizopogon-like type: single to pinnately branched or tuberculatemycorrhizae, white to light brown, rough mantle, abundant emanating hyphae without clamp connections, abundant strands formingmats; I,Cenococcum-like type: single mycorrhizae, rough and black, 1–3 mm long, black abundant emanating hyphae without clampconnections.

bLaccaria sporophores were found in a 10-cm circle around the core position.cNo sporophores were found in a 10-cm circle around the core position.dChalciporus piperatussporophores were found in a 10-cm circle around the core position.

Table 6. Mycorrhizal morphotypes collected in 17 soil cores from one plot inoculated byL. bicolor S238N in May1997.



1994). The absence of sporophores under control trees(Table 5) suggests that no naturally occurringLaccaria ge-nets were able to colonize them the year following out-planting; alternatively, they might not have enough access tohost photosynthetates to fruit (Lamhamedi et al. 1994), sug-gesting that they very recently established. This was ratherunexpected because of the presence of a rich natural ectomy-corrhizal microflora in the previous deciduous mixed forestthat was clearcut 1 year before planting Douglas-fir.

Eight to 12 years after outplanting, all the Douglas-firplots were colonized byL. laccata or L. bicolor strains(Table 6), as well as some other species, independently ofthe nursery treatments. Comparison of the aboveground(sporophore) and belowground (Table 6) surveys revealed adiscrepancy in the identified species, as described in otherstudies (Jansen and de Nie 1988; Mehmann et al. 1995;Gardes and Bruns 1996), but emphasized the dominance ofLaccaria spp. among the ectomycorrhizal community ofDouglas-fir plantation. In the natural forest surrounding theplots, we often foundL. laccata, but not L. bicolor sporo-phores (data not shown), a fact suggesting that species colo-nizing newly introduced Douglas-fir are recruited amongcommon and rare populations of the indigenous ectomy-corrhizal community. DNA typing ofLaccaria spp. sporo-phores confirmed that the two inoculated strains were stillpresent and dominant in the inoculated plots where they hadbeen introduced 10 years before. Because of the low ampli-fication rate of fungal rDNA from Douglas-fir mycorrhizae,we were not able to determine the degree of root coloniza-tion by the inoculated strains. Despite their abundant fruiting(also observed forL. bicolor S238N in nursery, (Henrion etal. 1994), they could be infrequent on roots. However, weconfirmed thatL. bicolor S238N was at least present on in-oculated Douglas-fir roots 12 years after nursery inoculationand 10 years after outplanting. S238N sporophores werepresent in all the buffer zones (Fig. 3, Table 2), suggestingthat this strain has formed mycorrhizae with the newlyformed roots of the noninoculated trees along the controlplots. The morphotyping (Table 6) needs to be refined by theuse of molecular tools (Mehmann et al. 1995; Gardes andBruns 1996) to ascertain the species and strains involved inthe Laccaria-like types, as well as the infection level of theinoculated strains. Unfortunately, RAPD genotyping cannotbe used for mycorrhizae that also contain plant DNA.

In the control and inoculated plots, the trees were alsoprogressively and more or less erratically colonized byindigenousL. laccata or L. bicolor strains (Fig. 3B). Thelimited extent of these natural genets suggests a recent es-tablishment, but their origin remains unclear. They couldoriginate from compatible basidiospores carried by the windfrom surrounding forest stands. We can exclude the survivalof vegetative mycelia or mycorrhizae from the previous for-est site at least forL. bicolor, which seems very rare in theindigenous forests (F. Le Tacon and M.-A. Selosse, unpub-lished data). The fact that the indigenousLaccaria genetsare not fragmented (Fig. 3B) and the lack of sporophores onnoninoculated plots 1 year after outplanting (Fig. 3A) alsosuggest that the indigenous genets established after outplanting.Among the 80 indigenousLaccaria genets recorded here, onlyone is fragmented (Selosse et al. 1998a, genet G). This genetmay come from the nursery, whereL. laccatagenets are regu-

larly found (Henrion et al. 1994). However, it was restrictedto one plot and was not scattered in all the experiment. Wecannot exclude a vegetative propagation along the newlyformed roots and a later fragmentation. The establishment ofthe other indigenous genets is likely to have arisen afteroutplanting, since no sporophores were recorded on non-inoculated plots 1 year after outplanting (Fig. 3A).

The presence of indigenous genets in the control plotsmay have prevented the vegetative colonization of the insideof the noninoculated plots (Table 2) by the two inoculatedstrains. They are likely to have been competitively blockedafter crossing the buffer zones. The fact that genet patchesof indigenousLaccariagenotypes rarely overlapped (Fig. 3B)also suggests that competition among genets delimited ex-clusive territories. However, at least one control plot wasinvaded by bothL. bicolor S238N and 81306 from neigh-bouring inoculated plots (Fig. 3B). The inside of this plotwas not colonized by naturally occurringLaccaria strains,contrarily to the others (Table 2, plot 6). We can speculatethat this absence of naturalLaccaria competitor allowed theinvasion of the inoculated strains by vegetative propagation.Other microbiological factors, such as other naturally occur-ring ectomycorrhizal fungi or bacterial populations (Garbayeand Duponnois 1992), could be involved.

This long-term study confirms the efficiency of the twoselectedL. bicolor strains on the growth of Douglas-fir, evenseveral years after outplanting. In their investigation ofeucalypts inoculated byHebeloma westraliensesp.nov. andSetchelliogastersp. in Australia, 1 year after outplanting,Thomson et al. (1996) reported a beneficial effect on treegrowth only in the cases where the inoculant species per-sisted. A similar relationship between persistence and hostresponse was found in a comparative analysis of variousinoculants of jack pine on oil-sands tailings up to 2 years af-ter inoculation (Danielson and Visser 1989). Combined per-sistence and growth improvement have also been obtainedup to 8 years after outplanting, withPisolithus tinctoriusonloblolly pine (Marx et al. 1988) andPaxillus involutusonoaks (Garbaye and Churin 1997). Persistence of inoculatedstrains may thus enhance the benefit of nursery inoculationand is a trait to be selected among potential ectomycorrhizalinoculants. Various factors also may affect this persistence,ranging from biotic factors, such as competition of localectomycorrhizal community (Buschena et al. 1992; Le Taconet al. 1992), to physical or chemical soil factors (Danielsonand Visser 1989; Thomson et al. 1992). TheL. bicolorstrains inoculated on Douglas-fir are well suited to brownsoils situated at medium altitude (300–800 m) (Le Taconet al. 1992)

We still do not know why these twoL. bicolor strains areso efficient at colonizing the roots and improving the growthof Douglas-fir or other forest species (Le Tacon et al. 1992).Genetic determinism of the host –L. bicolor strain compati-bility is unknown but probably polygenic (Kropp 1997). Di-rect nutritional effects are probably not solely involved inthe growth stimulation, since theL. bicolor strains used inthis study did not improve the concentration of mineral ele-ments in needles of Douglas-fir in nursery conditions(Table 4) or after field outplanting (F. Tacon and D.Bouchard, unpublished data), an observation also reportedby Danielson and Visser (1989) for various inoculants. In

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some experiments, ectomycorrhizal infection byL. bicoloreven decreases the internal concentration of total nitrogen incontainerized Douglas-fir seedlings (Gagnon et al. 1996; C.Karabaghli-Degron and F. Le Tacon, unpublished data).From recent studies (Karabaghli-Degron et al. 1998), it ap-pears that fungal auxins produced byL. bicolor S238Ncould, at least partly, stimulate aerial host growth. Whatcould be the reason of the efficiency of these twoL. bicolorstrains, our study demonstrates that they successfully sur-vived in inoculated plots at least 12 years after outplantingand vegetatively colonized the newly formed roots of theinoculated trees, despite the indigenous ectomycorrhizalinoculum. They also were able to spread out of the inocu-lated plots and vegetatively colonize some naturally mycor-rhizal trees. Nevertheless, the presence of these inoculatedstrains did not prevent the colonization of the Douglas-firroots by naturally occurring fungi, includingLaccaria spp.,and does not preclude the establishment of a diverse symbi-otic community. Although a clearer view of the undergroundpopulation and infection level is still required, the strain sur-vival and beneficial effects on the host clearly support theuse of nursery inoculation of selected ectomycorrhizalstrains for Douglas-fir plantation in Europe.

Acknowledgements

We thank the company France-Forêts, owner of the Saint-Brisson stand, for access to the experimental plots. Thiswork was supported by a grant (59226) of the Bureau desressources génétiques and a E.U. contract (PL 931742). M.-A.S. is on leave from the École nationale du génie rural, deseaux et des forêts.

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Effect of Laccaria bicolor strains inoculated on Douglas-fir ( Pseudotsuga menziesii ) several years after nursery inoculation