Applied Soil Ecology 15 (2000) 125–136

In vitro and post vitro inoculation of micropropagatedRhododendrons with ericoid mycorrhizal fungi

Jan Jansa1, Miroslav Vosátka∗Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pruhonice, Czech Republic

Received 31 May 1999; received in revised form 25 November 1999; accepted 23 March 2000

Abstract

Isolation of more than 200 strains of endophytic fungi from the roots of several host plants belonging to orderEricales(Vaccinium, Calluna, Rhododendron, Empetrum, etc.) was followed by a successful attempt to verify ericoid mycorrhiza statusof some of these fungal isolates under axenic conditions. In two screening experiments, the most efficient ericoid mycorrhizafungal strains were found beneficial for the growth of micropropagated Rhododendron plants when inoculated post vitroafter transplantation to peat-based substrate. No negative influence on the growth of host plants has been observed for anyinoculated isolate, while about 10% of tested strains exhibited positive effects on the growth of Rhododendron microcuttingsgrown in peat-based media. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Ericoid mycorrhiza; Ericaceae; Horticulture; Micropropagation

1. Introduction

Ericoid mycorrhiza (ERM) belong, together withorchidaceous and arbuscular mycorrhiza types, to thegroup of endotrophic mycorrhizal associations. Eri-coid mycorrhizas are associations between ascomyce-tous (or rarely hyphomycetous) fungi and plant speciesbelonging to the familiesEricaceae, EpacridaceaeandEmpetraceae(Smith and Read, 1997). Also arbutoidand monotropoid mycorrhiza can be found within thefamily Ericaceaebut the most common ericoid my-corrhiza are found in genera such asCalluna, Erica,

∗ Corresponding author. Tel./fax:+42-2-67750022.E-mail addresses:jan.jansa@ipw.agrl.ethz.ch (J. Jansa),vosatka@ibot.cas.cz (M. Vosatka)

1 Present address: IPW-ETH Zürich, Eschikon 33, Postfach 185,CH-8315 Lindau (ZH), Switzerland. Tel.:+41-52-3549216;fax: +41-52-3549119.

Rhododendron, Vaccinium, andEmpetrum(Smith andRead, 1997).

The ERM is characterized by considerably uniformstructure, similar to those in arbuscular mycorrhizas,but usually more delicate (Peterson et al., 1980; All-away and Ashford, 1996). A range of differently sep-tated hyphae (simple septal pores of ascomycete typeas well as dolipore septated fungi of basidiomyce-tous type can occur) were found inside the corti-cal cells of ericaceous plants (Bonfante-Fasolo andGianinazzi-Pearson, 1979; Bonfante-Fasolo, 1980).The hyphae of ERM fungi penetrate a single layer ofcortical cells of the roots and fill them with intracel-lular hyphal coils. Cortical cells of ericaceous plantsnever form structures like root-hairs, well describedin other plant families.

The ericaceous plants occur in most climaticallyand edaphically stressed environments particularlywhen soil acidity becomes extreme and the rate of

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nutrient mineralization is low. The function of theERM fungi is, most probably, to cover nutrient de-mands of the plant under such stress conditions(Harley, 1969). The ERM fungi in acidic heath soilsproduce external mycelium that is supposed to havean active function in obtaining mineral nutrients: en-zymatic release of nitrogen from predominant organiccompounds, that would otherwise be unavailable forthe roots. Most remarkable is a high C:N ratio inthese soils, which is overcome due to the activity ofenzymes produced by the ERM fungi. The ericoidmycorrhizal plants showed access to nitrogen sourcesthat are almost inaccessible for nonmycorrhizal plants(Pearson and Read, 1975; Kerley and Read, 1998). En-zymes hydrolyzing different carbon polymers (oligo-and polysaccharides, including cellulose and pectins)were described from pure cultures of the ERM fungi(Pearson and Read, 1975; Perotto et al., 1993; Varmaand Bonfante, 1994). Effective uptake of nitrogen bythe ERM fungi from different sources in peat-basedsoils was described — both free ammonia and nitratefixing pathways are more efficient for fungi comparedto the plants, especially under acidic conditions (Pear-son and Read, 1975). Organic polymers-boundednitrogen was utilized by the ERM fungi especially un-der low pH (Leake and Read, 1990a) and chitinolyticactivity of the fungi was proved as well (Leake andRead, 1990b). Increase in phosphate uptake of erica-ceous host plants due to ERM fungi was described byRead and Stribley (1973), mainly due to solubilizationof ferric or aluminium phytates (Mitchell and Read,1981). Moreover, a phosphodiesterase attacking nu-cleic acid bound phosphate was described from fungalcultures by Leake and Miles (1996). A high affinityof the ERM fungi for iron is probably important asmaintained by production of siderophores (Schulerand Haselwandter, 1988). Some of the ericoid my-corrhizal host plants (Calluna and Vaccinium) werereported for high tolerance to different environmen-tal stresses. This makes these ERM fungi practicallyinteresting for their potential to enhance plant fitnessunder unfavourable conditions (Bradley et al., 1982;Burt et al., 1986; Yang and Goulart, 1997). Attemptswere made to find a proper fungal strain to decreaselosses in propagation of Rhododendrons at commer-cial level, which may reach up to 10% at weaningstage (Lemoine et al., 1992). This work showed com-plexity and probable strain-to-strain specificity of

plant and fungi with respect to positive plant growthreaction, which may depend also on substrate type.

The ERM fungi are characterized by a very slowdevelopment — first apparent colonization struc-tures were observable after 3 weeks ing-irradiatedreinoculated soil and after 4 weeks in horticulturalsoil, collected under Rhododendrons (Duddridgeand Read, 1982). This documents possible role ofother microorganisms, which may slow down thedevelopment of the fungus to a certain extent. Thebreakdown of some of the mycorrhizal structures wasevident after 8 or 11 weeks in irradiated or unsterilesoil, respectively. The breakdown process starts bystructural desintegration of plant organelles and cells,followed by a loss of integrity of the fungal struc-tures (Duddridge and Read, 1982). This means thatthe fungus at least in part of its life span plays a roleof a sapro-parasitic partner. Simply septated fungi,supposed to be symbiotic ones, frequently observedwithin cortical cells, can be isolated from roots, butthey very scarcely form spores in culture, which hin-ders their classification. They are divided into twogroups — slow growing, dark-coloured and usuallysterile mycelia (McNabb, 1961; Pearson and Read,1973a; Singh, 1974), and a group ofOidiodendronsp. observed mainly in isolations fromCallunaor Vac-cinium(Couture et al., 1983; Dalpe, 1986; Douglas etal., 1989). Considerable genetic diversity was foundamongst isolates that were superficially very similar,on both biochemical-isozymes (Hutton et al., 1994)and molecular basis (Perotto et al., 1995), as well asclassical microbiological techniques (Hambleton andCurrah, 1997). A question remains as to whether suchdiversity observed in root-associated fungi has anyimpact upon performance of plants grown under con-trolled conditions (Smith and Read, 1997). A demandto extend the research observation beyond classicalCalluna–Vaccinium–Rhododendronmodel to includemore taxonomically diverse host and fungal partnershas arisen in recent time (Straker, 1996).

The objective of our work was to isolate endophyticfungi from different plants and to attempt the reinoc-ulation of micropropagated Rhododendron plantlets.This was made in both in vitro system, accord-ing to Koch’s postulate (Koch, 1912), to revealmycorrhizal status of these fungi, and in post vitrosystem, to check for any growth effect of mycorrhizaon plants.

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2. Material and methods

2.1. Isolation of ericoid mycorrhizal fungi

The roots of different ericaceous plants weresampled in nature from a range of habitats (fromRhododendrons cultivated in botanical gardens toVaccinium grown in high altitude mountain area).The roots were stained to confirm presence of fun-gal structures according to the following protocol:maceration of the roots in 10% KOH in autoclave at121◦C for 1 h, cleaning with 5% hydrogen peroxidefor 15 min, washing under tap water, acidifying with1% HCl and staining for 2 h in 0.1% Trypan Blue inlactoglycerol (lactic acid:glycerol:water — 1:1:1) at80◦C and leaving the roots in the staining solutionat room temperature overnight. After this, the rootswere destained in water for 24 h and observed undercompound microscope at the magnification of 400×.

The technique for isolation of the ERM fungi fromroot samples was modified from Pearson and Read(1973a). The root samples were washed with tapwater and surface sterilized by six times repeatedlyshaking in sterile tap water (to protect plasmolysis ofthe fungal hyphae, which usually occurs while usingdistilled water). This was followed by sterilizationstep with active chlorine. The roots were submergedin 5% solution of SAVO bleaching solution (10%NaOCl) for 3 min, then the roots were transferred into5% hydrogen peroxide after washing the previoussolution out, followed by another 5 min in 5% SAVObleaching solution. All the sterilization solutions usedcontained a drop of detergent (BRIJ 35) to supportappropriate wetting of the root surface. Roots werecut under sterile conditions using scissors into piecesapproximately 5 mm long and they were put onto thesurface of the isolation medium containing strepto-mycin, to prevent the growth of bacteria. Sixteen rootpieces were equally distributed over the surface of9 cm Petri dishes. Root segments were cultivated for3 weeks on both nutrient (1% malt extract) and wateragar at pH 4.5 (according to Leake and Miles, 1996)at room temperature (25◦C) in dark.

2.2. Cultivation and maintenance of ericoidmycorrhizal fungi

Particular isolates of the ERM fungi growing fromthe roots were transferred on the specific medium

after Dalpe (1990) and on malt extract agar (MEA).The cultures of isolated ERM fungi strains were main-tained on the medium in both Petri dishes and vialswith skewed agar plates. The vials were closed with aParafilm foil, sealed over the stopper, to ensure goodviability of the isolates cultures (up to 1 year) understorage at 4◦C. For preparing inocula of ERM fungi,liquid cultures were used. Ten 9 cm Petri dishes con-taining 25 ml of cultivation media (4% malt extract,pH 4.5) were used for each isolate. The cultures wereleft to grow for 4 weeks in dark at 20◦C. Then themycelia were collected on a paper filter, homogenizedby blending at high speed by Waring blender in 200 mlsterile water for 5 s and diluted in sterile water to finalvolume of 500 ml.

2.3. Inoculation in vitro

Experiment 1: Azalea cv. AK 504 were grown invitro on modified 0.5% agar medium after Anderson(1984) without phytohormones, amended with 2 g ofcharcoal per 1l of media, pH 5.2. Re-establishmentof mycorrhiza was performed in sterile 200 ml Erlen-meyer flasks, filled with 30 ml of Lignocel substrate,enriched with 12 g Osmocote per 1 l. Inoculation wasmade using mycelium plugs from vial cultures af-ter 3 weeks acclimation period following transfer ofplantlets from nutrient medium. Ten strains of theERM fungi were used for inoculation and the plantswere grown for 12 weeks. For evaluation of presenceof mycorrhizal structures, plant roots were processedas described above.

2.4. Inoculation experiments, post vitro

Experiment 2: In vitro propagated seedlings ofRhododendron cv. Belle-Heller were used in postvitro inoculation experiments. Regenerants fromstem-induction medium were transplanted onto root-ing medium (Lignocel peat, prepared from tree bark,enriched with coconut fiber: 500 g of Lignocel per4.5 l of distilled water — pH 4.8) amended with3 g of Osmocote slow release fertilizer (5–6 monthsrelease time) per 1 l of substrate. After 8 weeks,plantlets were transplanted into 3.5 l plastic boxeswith Lignocel enriched with 3 g Osmocote per 1 l.For inoculation of one plant, about 1.3 mg dry weightof mycelia was used, pipetted under each plant. Also

128 J. Jansa, M. Vosatka / Applied Soil Ecology 15 (2000) 125–136

the root system of the seedlings was dipped into themycelial suspension before planting into inoculatedcontainer. Each experimental container represented aunit infected with one of the 12 selected ERM fun-gal isolates; 15 plants were grown in each container.Mortality, biomass of shoots and roots, leaf area

Fig. 1. (a) Intraradical colonization ofCalluna vulgarisfrom natural habitat (Jesenıky Mts.) by an ericoid mycorrhizal fungus (bar represents10mm). (b) Detail of hyphal coils of an ericoid mycorrhizal fungus within cortical cells of a root ofRhododendronsp. from natural habitat(Jesenıky Mts.) (bar represents 10mm).

were evaluated after 12 weeks of cultivation in thegreenhouse.

Experiment 3: This experiment was set in a similarway as the previous one except that 20 different strainsof ericoid mycorrhiza isolates were used for inocula-tion. In this case, the cultivation period was 16 weeks.

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2.5. Statistical evaluation of the data

One-way ANOVA was used for evaluation of theeffect of different fungal isolates on plant growth.G-test was used only to estimate significant differencesin mortality rates. The statistical evaluation was per-formed using SOLO statistical package (BMDP Soft-ware, Los Angeles, CA, 1991).G-test was calculatedusing CoStat package.

3. Results

Almost all roots of ericoid plants collected from thenatural habitats were found to have some kind of in-timate hyphal association within cortical cells of theirroots (Fig. 1a and b). Mycorrhizal colonization waswell developed in the whole range of habitats and alti-tudes (from 400 to 1200 m above sea level). High col-onization rate of roots sampled in nature was detected(different cultivars ofRhododendronsp., Vaccinium

Table 1Isolates of ericoid mycorrhizal fungi used for experimental work and their origin

Isolate Description Host plant and origin

O1 Oidiodendronsp. Rhododendronsp., 100 years old, PruhoniceO2 Oidiodendronsp. Rhododendronsp., 100 years old, PruhoniceO3 Oidiodendronsp. Vaccinium myrtillus, K. StudankaO4 Oidiodendronsp. Vaccinium myrtillus, K. StudankaO5 Oidiodendronsp. Rhododendronsp., 120 years old, PruhoniceO6 Oidiodendronsp. Rhododendronsp., 40 years old, PruhoniceO7 Oidiodendronsp. Vaccinium myrtillus, Jesenıky Mts.O8 Oidiodendronsp. Vaccinium myrtillus, Jesenıky Mts.O9 Oidiodendronsp. Empetrum hermaphroditum, Oulu, FinlandO10 Oidiodendronsp. Rhododendron, 100 years old, PruhoniceO11 Oidiodendronsp. Rhododendron, 40 years old, PruhoniceD1 Dark sterile mycelium Vaccinium myrtillus, Oulu, FinlandD2 Dark sterile mycelium Vaccinium myrtillus, Oulu, FinlandD3 Dark sterile mycelium Empetrumsp., Oulu, FinlandD4 Dark sterile mycelium Loisaleuria procumbens, UkraineD5 Dark sterile mycelium Rhododendronsp., Karlova StudankaD6 Dark sterile mycelium Vaccinium myrtillus, Jesenıky Mts.D7 Dark sterile mycelium Rhododendronsp., 30 years old, PruhoniceD8 Dark sterile mycelium Gaultheria procumbens, UkraineD9 Dark sterile mycelium Rhododendronsp., 30 years old, Pruhonice,D10 Dark sterile mycelium Rhododendronsp., 100 years old, PruhoniceD11 Dark sterile mycelium Vaccinium myrtillus, Karlova StudankaD12 Dark sterile mycelium Vaccinium myrtillus, Karlova StudankaD13 Dark sterile mycelium Rhododendronsp., 100 years old, PruhoniceD14 Dark sterile mycelium Vaccinium myrtillus, Karlova StudankaD15 Dark sterile mycelium Rhododendronsp., 100 years old, Pruhonice

myrtillus, Calluna vulgaris, Gaultheria procumbens,andEmpetrum hermaphroditum).

More than 200 strains of endophytic fungi belong-ing to Oidiodendronsp. and dark sterile (ascomyce-tous) mycelia were isolated from the roots of differenthost plants (Table 1). Satisfactory growth was obtainedon MEA medium, of slowly growing, dark and sterilemycelia, revealed to develop from hyphal coils insidecortical cells and were supposed to be the symbioticfungi. During their growth, they were usually maskedby rapidly growing saprophytes.

Using our isolation technique, a trend was observedin distribution of symbionts amongst different hostplants, a higher number ofOidiodendronsp. were iso-lated fromVacciniumplants than from Rhododendrons(Table 2). Majority of root-endophytic fungal popu-lations by other plants make fungi belonging to darksterile mycelium group.

In vitro mycorrhization of Azalea plantlets (exper-iment 1) showed stimulation of rooting byOidioden-dron (strains O4 and O6). Fungal isolates inoculated

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Table 2Numbers of morphologically different colonies of fungal endo-phytes obtained by isolation from root samples ofCalluna vul-garis, different Rhododendrons, Vaccinium myrtillus, Gaultheriaprocumbensand Empetrum hermaphroditumcollected in naturalhabitats

Plant sample Dark sterilemycelium

Oidiodendronsp.

Calluna 3 0Rhododendron 1 14 2Rhododendron 2 12 2Rhododendron 3 15 0Rhododendron 4 8 2Rhododendron 5 3 0Rhododendron 6 6 4Rhododendron 7 13 0Vaccinium 1 4 6Vaccinium 2 4 5Vaccinium 3 4 7Vaccinium 4 6 2Vaccinium 5 3 3Gaultheria 10 0Empetrum 1 2 2Empetrum 2 5 0

into the system developed structures typical of ericoidmycorrhizas (Fig. 2a and b). However, the majorityof fungi appeared to grow too fast in relation to theplants and after harvest, inhibitory effect of fungalgrowth on root development was observed. Thereappeared to be a competition for mineral sources, asthe plants were showing symptoms of deficiencies.In prolonged cultivation, after another 4 weeks, theplants were overgrown by fungi. Not enough rootsfor evaluation of infection were obtained, thereforeonly presence–absence of mycorrhizal structures wererecorded (Table 3).

Post vitro mycorrhization of Rhododendrons in ex-periment 2 showed that there was only one isolate (D4)significantly stimulating plant growth. However, therewere no inhibitiory effects of any inoculated fungus(Table 4, Fig. 3). Similarly, in experiment 3, two (O6,O9) of 20 strains of ERM showed consistent positiveeffects on plant growth (Table 5, Fig. 4). Root biomasswas generally reduced by inoculation in experiment2, but such an effect was not observed in experiment3, probably due to prolonged cultivation time. Mostapparent stimulation of plant development due to in-oculation was observed using isolate O6 in both post

vitro experiments (significant leaf number increase inexperiment 2, significant stem dry weight and leaf areastimulation in experiment 3). Increase in some of theparameters describing plant growth were also observedfor fungal isolate D6 in both experiments.

4. Discussion

The abundance of ERM fungi in the roots of the ma-jority of plants sampled indicates the wide ecologicalimportance of the symbiosis (Haselwandter, 1979).Isolated strains of ericoid mycorrhizal fungi wereproved to be true endophytic symbionts and formingericoid mycorrhizas with tested plants. Soil-wateragar, used for axenic cultures establishment by Pear-son and Read (1973a), and Read and Stribley (1973)has the advantage of slowing growth of the fungus,while rich substrates, as that used in our work, havethe advantage of supporting growth of the plant fora longer time. During longer periods, a better devel-opment of mycorrhizal structures may be achieved.On the other hand, the cultivation substrate shouldbe probably first inoculated with some bacteria thatwould diminish the amount of easily available nutri-ents which are released during sterilization process(autoclaving), causing a too fast growth of the fungusrelative to the development of the plant. However,this makes the system less convenient for assessingthe influence of the fungus on the plant growth, as itscomplexity increases. On the other hand, using thissystem we may get near to the real state, compared tousing extremely poor environment of water or weaksoil-water agar (which are the generally used ones fortesting mycorrhization capabilities of fungal strainsin vitro, Pearson and Read, 1973a,b). Rich-substratetechnique, described in this work, may also be readilyexploited in biotechnological application in indus-trial production, just before ex vitro transplantationof plantlets (which have to be kept on rich media toensure good survival after).

Autoclaving as the technique of sterilization usedin this work may change the nutrient and toxin con-tent of the substrate. According to one of the earlytheories of functioning of the ERM, the fungi serve asdetoxifying agents in the substrate (Freisleben, 1935).The influence of the ERM fungi on vitality and growthof plants seems to be a combination between the

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Fig. 2. (a) Detail of hyphal coil in the cortical cell ofAzalea root inoculated in vitro with an ericoid mycorrhizal fungus (isolate D5)isolated fromRhododendronsp. from natural habitat (Jesenıky Mts.) (bar represents 10mm). (b) Detail of intraradical hyphae — pelotonsof an ericoid mycorrhizal fungus (isolate D5) within root ofAzaleainoculated in vitro with an ericoid mycorrhizal fungus isolated fromRhododendronsp. from natural habitat (Jesenıky Mts.) (bar represents 10mm).

detoxifying role and the role of host nutrition (Smithand Read, 1997). In spite of these complications,steam sterilized sand or soil mixtures are the most fre-quently used substrates for glasshouse experiments,being reliable concerning the growth response ofplants to inoculation (Stribley and Read, 1976).

The increase in growth of plants after inoculationof some ERM fungi in our experiments correspondsto the results of Pearson and Read (1973a), Read andStribley (1973) or Stribley and Read (1974), whoalso found stimulation of plant growth, measured asbiomass of shoots and roots. Very similar results were

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Table 3Mycorrhizal status of Azaleas, in vitro inoculated with 10 different fungal endophytes isolated from the roots of ericoid plants [experiment 1]

Treatment (ERM isolate) Description Mycorrhizal status

Control No colonization, no intracellular structures –a

D1 Heavy colonization by thick hyphae +b

D2 Thick hyphae, roots covered by hyphae, intracellular pelotons +b

D4 Typical dark mycelium colonizing roots, pelotons! ++c

D5 Superficial hyphal mantle, hyphae penetrating into inner cell layers +b

D7 Fine, dark mycelium ++c

D8 Superficial hyphal mantle, dark pelotons inside cells ++c

O4 Nonspecific intracellular colonization, fine mycelium tomassive intracellular infection, no pelotons

+b

O6 Bright mycelium, heavy colonization ++c

O7 Fine intracellular colonization, pelotons +b

O8 Nonspecific hyphae ?d

a No mycorrhiza.b Mycorrhizal fungus.c Mycorrhizal fungus of high infectivity.d Uncertain mycorrhizal status.

Table 4The effect of different isolates of ericoid mycorrhizal fungi on the mortality and growth of Rhododendrons inoculated post vitro (G-testincluding Williams correction at 5%)a [experiment 2]

Treatment(ERM isolate)

Mortality (%) significantdifference vs control

Shoot dryweight (g)

Root freshweight (g)

Leaf area(cm2)

Leafnumber

Control 13 0.19 b 1.16 a 27.1 b 10.3 bO1 30 ns 0.23 b 0.88 b 35.5 b 16.9 abO2 20 ns 0.09 b 0.31 b 11.6 b 9.8 bO3 20 ns 0.24 b 0.67 b 39.3 b 12.6 bO4 50 ns 0.19 b 0.63 b 29.8 b 9.8 bO5 70∗ 0.20 b 0.66 b 32.0 b 11.4 bO6 30 ns 0.30 ab 0.84 b 44.5 ab 17.7 aD1 27 ns 0.16 b 0.41 b 24.0 b 10.0 bD2 27 ns 0.18 b 0.49 b 27.1 b 11.7 bD3 40 ns 0.27 b 0.75 b 42.6 b 12.3 bD4 7 ns 0.40 a 1.02 ab 64.5 a 15.3 bD5 27 ns 0.25 b 0.58 b 36.0 b 9.7 bD6 13 ns 0.31 ab 0.75b 53.5 ab 12.6 b

∗ Treatment is significantly different from control.a Duncan’s Multiple range testp<0.05, means followed by the same letter are not significantly different within one column.

also reached by Lemoine et al. (1992), who reportedgrowth stimulation by Rhododendron microcuttingsdue to inoculation with symbiotic fungus up to 200%of the growth of the control plants, together withdecreased heterogeneity of inoculated plants. Con-trary to our findings, a positive influence of the ERMfungi on survival of the host plants was observed byNieuwdorp (1969) and Lemoine et al. (1992). Suchdiscrepancy may be caused by different host plantsand experimental conditions, even if close species

to species specificity of ericoid symbionts was notproved (Douglas et al., 1989). However, some prefer-ences in specificity were observed by Xiao and Berch(1995) and also Lemoine et al. (1992). Stribley et al.(1975) have measured influence of the ERM fungi onthe host plant also by relative growth rate and specificabsorption rate of nitrogen. Significant increase inroot and shoot biomass, due to inoculation withPez-izella ericaeunder axenic conditions was observedby Berta and Gianinazzi-Pearson (1986) after 1 and

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Fig. 3. The effect of different isolates of ericoid mycorrhizal fungi on the growth of Rhododendrons inoculated post vitro, (normalizeddata from experiment 2 — normalization by division by the mean value), (*) significant increase (5% level) due to inoculation with thefungus, compared to the control.

4 weeks, respectively. Also a significant shift in rootarchitecture due to ERM fungi corresponding to thelevel of endogenous auxins was described by Bertaet al. (1988). That may be an indication of the cause ofsome of the reported morphological and physiologicaleffects. Positive influence of the presence ofPezizellaericaewithin the roots ofVaccinium corymbosumon

Table 5The effect of different isolates of ericoid mycorrhizal fungi on the growth of Rhododendrons inoculated post vitro (means followed by thesame letter are not significantly different within one column according to Duncan’s Multiple range testp<0.05) [experiment 3]

Treatment (ERM isolate) Shoot dry weight (g) Root fresh weight (g) Leaf area (cm2) Leaf number

Control 0.72 cdef 3.68 bcd 110.7 cd 13.5 bcD1 0.75 cde 3.68 bcd 127.0 cd 16.3 bcD10 0.55 def 2.81 cde 81.6 def 14.5 bcD11 0.77 bcde 3.62 cd 119.3 cd 14.2 bcD12 0.77 bcde 3.96 bc 141.7 bcd 17.9 bcD13 0.68 cdef 3.29 cde 106.4 cd 15.7 bcD14 0.64 cdef 3.36 cde 110.8 cd 15.9 bcD15 0.70 cdef 3.62 cd 114.3 cd 17.0 bcD2 0.81 bcd 3.80 bc 136.0 bcd 16.8 bcD4 0.74 cde 4.02 bc 128.2 cd 16.4 bcD6 1.07 abc 5.02 abc 146.8 bcd 14.9 bcD7 0.98 abcd 4.77 bc 150.7 bcd 30.2 aD8 0.80 bcde 4.02 bc 134.2 bcd 16.1 bcD9 0.75 cde 3.96 bc 131.0 cd 18.1 bcO10 0.59 cdef 2.94 cde 96.2 cde 16.1 bcO11 0.83 bcd 4.33 bc 130.1 cd 14.4 bcO3 0.68 cdef 3.39 cde 118.0 cd 15.5 bcO6 1.24 ab 6.07 ab 200.6 ab 20.9 bO7 0.96 abcd 5.06 abc 160.9 abc 17.6 bcO8 0.66 cdef 3.43 cde 116.4 cd 17.9 bcO9 1.39 a 7.17 a 216.4 a 15.9 bc

the growth and nutrient uptake by the host plants wasalso described by Powell (1982), and forCallunavulgarisby Strandberg and Johansson (1999).

Our results indicate that different fungal isolateseven if isolated from one host plant, being almost im-possible to distinguish, may have a different influenceon the growth of the host plants artificially inoculated.

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Fig. 4. The effect of different isolates of ericoid mycorrhizal fungi on the growth of Rhododendrons inoculated post vitro (normalizeddata from experiment 3 — normalization by division by the mean value), (*) significant increase (5% level) due to inoculation with thefungus, compared to the control.

Such a behaviour was hypothesized previously (Smithand Read, 1997), but only a little information wasavailable till now (Douglas et al., 1989). Very specificenzymatic apparatus of the ERM fungi was described,releasing nutrients only from a certain spectra ofsource molecules, that is supposed to vary, dependingon isolates and certain conditions (Bajwa and Read,1986). That may explain, why some ERM fungal iso-lates may not demonstrate their beneficial effects onthe growth of the host, according to the conditions,under which they occur. We have described beneficialinfluence of inoculation with ERM fungi only in about10–15% of isolates, which corresponds to the resultsof Gianinazzi-Pearson (pers. commun.). However,since some of the ERM fungi strains were proved notto stimulate plant growth, selection for appropriatestimulatory strains for practical inoculation is essen-tial. Not regarding the principles of functioning of theericoid mycorrhiza, the consistent improvement of thegrowth of ericaceous plants cultivated under artificialconditions described in this work may contribute tohorticultural practices. The selection of isolates withhighest stimulative capabilities in a given environ-ment is needed (especially regarding substrate typeand pH as well as host cultivar identity), as alreadyproposed by Lemoine et al. (1992).

Further research on selected strains should continueto find appropriate doses, survival potential of my-corrhiza in the substrate and the most efficient way ofinoculum introduction into the substrate. That could

lead to exploitation of these fungi for inoculation ofericaceous plants in horticultural practices.

Acknowledgements

We would like to express many thanks to Mrs. JanaRouckova for her outstanding technical assistance, aswell as to other members of Mycorrhizal Group inthe Institute of Botany, Pruhonice for their kind helpand to Ministry of Education, Youth and Sports of theCzech Republic for financing cooperative grant ME256 and to the colleagues from University of Ljubl-jana, Slovenia for valuable cooperation.

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