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Mycorrhiza (1999) 8 :271276 Q Springer-Verlag 1999

ORIGINAL PAPER

Ana Rincn 7 Isabel F. lvarez 7 Joan Pera

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

Accepted: 29 October 1998

A. Rincn 7 I.F. lvarez 7 J. Pera (Y)

Departament de Patologia Vegetal,Institut de Recerca i Tecnologia Agroalimentries (IRTA),Centre de Cabrils, Ctra. de Cabrils s/n,E-08348 Cabrils, Barcelona, SpainFax:c34-93753 3954, e-mail: joan.pera6irta.es

Abstract Although Pinus pinea L. is an important for-est species in the Mediterranean region, few reports ex-ist on its ectomycorrhizal associates. Sixty isolates, ob-tained from fungal sporocarps collected in mixed for-ests of P. pinea in Catalonia (northeastern Spain), weretested for ectomycorrhiza formation on containerizedP. pinea seedlings when applied as mycelial inoculumproduced in peat-vermiculite. A total of 17 isolates, in 8genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisol-ithus, Rhizopogon, Scleroderma and Suillus), formedectomycorrhizas and the percentages of mycorrhizalshort roots varied among isolates and species from 13%to 89%. Some of these fungi are cited for the first time

in association with P. pinea. The results indicate furtherfungal candidates for controlled inoculation of P. pineaseedlings in the nursery.

Key words Stone pine 7 Synthesis of mycorrhizas 7Containerized seedlings 7 Mycelial inoculum producedin peat-vermiculite

Introduction

Pinus pinea L. is one of the most representative conifer

species of the Mediterranean region. It extends fromPortugal to the Syrian and Lebanese coastal areas, al-though 75% of its world-wide distribution is located inthe Iberian peninsula (Agrimi and Ciancio 1994). It to-lerates the hot and dry conditions associated with theMediterranean climate and occurs in a wide range ofsoils and sites, but is usually found in infertile, sandysoils unfavourable for other forest species (Montoya

1989). Pinus pinea is mainly valued for its edible nutsand it plays an important ecological role in arid andsemi-arid zones by preventing erosion (Montero et al.1997). This species is thus useful in standard reforesta-tion and the afforestation of marginally economic agri-cultural lands.

Different species of Pinus have been used frequentlyin reforestation programs around the world (Castellano1994, 1996). The association of plant roots with ectomy-corrhizal (EM) fungi is an important factor in the per-formance of outplanted conifers (Marx 1991; Castella-no 1996) and it has been demostrated that Pinus spp.are dependent on the symbiosis to reach optimal devel-

opment under natural conditions (Marx 1980; Molina etal. 1992). The application of controlled inoculationtechniques to nursery-grown seedlings can be a usefultool for enhancing field performance of outplanted see-dlings (Cordell et al. 1987). However, not all fungi pro-duce the same effects on a particular tree species andvarious criteria must be taken into account in the selec-tion of the most effective EM fungi in a nursery inocu-lation program (Trappe 1977; Marx 1980; Molina andTrappe 1984).

For selection of appropiate fungi, the fungal sym-bionts of the plant species must first be defined. Var-

ious methods have been developed for determining theability of a fungus to form ectomycorrhizas with a par-ticular tree species (Melin 1922; Hacskaylo 1953; Marxand Zak 1965; Fortin et al. 1980; Molina and Palmer1982; Duddridge 1986; Stenstrm and Unestam 1987;Wong and Fortin 1989). Most require axenic conditionsbut non-axenic methods have also been used (Marx1976; Danielson et al. 1984; Dunabeitia et al. 1996).

Cenococcum geophilum Fr. (Trappe 1962), Hebelo-ma crustuliniforme (Bull. ex St. Am.) Qul. (Branzantiet al. 1985), Hebeloma sinapizans (Paul. ex Fr.) Gillet(Branzanti and Zambonelli 1988), Laccaria laccata

(Scop. ex Fr.) Bk. & Br. (Branzanti et al. 1985; Bran-zanti and Zambonelli 1988), Lactarius deliciosus (L. exFr.) S. F. Gray (Trappe 1962), Lactarius hygrophor-oides Berk & Curt. (Barsali 1922), Paxillus involutus


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(Batsch. ex Fr.) Fr. (Branzanti and Zambonelli 1988),Pisolithus tinctorius (Pers.) Cok. & Couch (Mousain etal. 1987, 1994), Rhizopogon roseolus (Corda ex Sturm)Fries (Mousain et al. 1987, 1994), Suillus collinitus (Fr.)O. Kuntze (Mousain et al. 1987; 1994), Suillus granula-tus (L. ex Fr.) O. Kuntze (Takacs 1961; Branzanti andZambonelli 1988), Suillus luteus (L. ex Fr.) S. F. Gray(Trappe 1962), Tuber albidum Pico (Branzanti andZambonelli 1984; Pascualini et al. 1992) and Tuber bor-chii Vitt. (Barsali 1922; Ceruti 1965) have been cited inassociation with P. pinea.

Research is currently under way, mainly in Franceand Spain, to select EM fungi to improve field perform-ance of outplanted P. pinea seedlings. This presentwork is an initial step to increase current knowledge ofEM fungi associated to P. pinea in order to develope aselection process for their use in nursery inoculationprograms.

Materials and methods

Sporocarps of putative EM fungi were collected throughout theyear in mixed forest of P. pinea in diverse locations of Catalonia(northeastern Spain). Site characteristics were recarded. Isolationand taxonomic identification were carried out on the differentsporocarps collected. To obtain pure culture isolates, the sporo-carps were externally brushed and pieces of inner tissue were tak-en and placed in Petri dishes filled with modified Melin-Norkransagar medium (MMN) (Marx 1969). Cultures were incubated for730 days at 25 7C, depending on the species. The isolates ob-tained were transferred to MMN-filled tubes and maintained by

transfer on MMN every 3 months (Molina and Palmer 1982;Smith and Onions 1994).Pinus pinea seeds were obtained from natural forests in the

Montnegre and Montseny Sierras in Catalonia. Seeds were rinsedovernight in running tap water, surface disinfected by shaking for60 min in 30% H2O2, and washed in two changes of sterile dis-tilled water. Disinfected seeds were sown on trays (10 seeds pertray) filled with wet, sterilized perlite (30 min, 120 7C), and germi-nated in a greenhouse under controlled temperature (1825 7C)and humidity conditions (140%).

The synthesis of ectomycorrhizas was carried out on contai-nerized seedlings under greenhouse conditions, using vegetativemycelial inocula of 60 different fungal isolates (Table 1). To ob-tain the vegetative inoculum, each fungal isolate was grown inMMN liquid medium (25 7C, 715 days) and then 4050 ml of the

mycelial suspension was transferred to 1-l bottles containing anautoclaved (20 min, 120 7C) mixture of peat-vermiculite(50550 ml) moistened with 350 ml of MMN liquid medium (glu-cose reduced to 2.5 g/l). Inoculated bottles were incubated at25 7C in the dark for 1 month. The peat-vermiculite inoculum wasmixed with sterilized substrate (peat-vermiculite, 1: 1, v:v; pH 5.5)in the proportion 1:4 (inoculum:substrate by volume). EmergentP. pinea seedlings were transferred from germination trays to 175-ml containers (Sherwood, Rootrainers, Spencer-Lemaire) filledwith the inoculated substrate. A total of 14 plants were inoculatedwith each fungal isolate tested. The inoculated plants were grownin a shaded greenhouse with a controlled enviroment (2025 7Cand 140% humidity) for 4 months. At the end of this period,plants were removed from the containers and their root systemswashed prior to examination for the presence of ectomycorrhizas.

To assess the percentage of colonized short roots, at least 200 ran-domly selected short roots of each plant were counted under thestereomicroscope. Samples of infected roots from each seedlingwere sectioned transversely for microscopic examination of Har-tig net formation.

Results

A total of 289 sporocarps (80 species in 18 genera) ofputative EM fungi were collected and 109 isolates ob-tained in pure culture. Fungi belonging to the generaBoletus, Lactarius, Pisolithus, Rhizopogon and Suillus

were easily isolated in pure culture (in more than 50%of the cases), in contrast to species of Laccaria and Rus-sula, which were difficult to isolate from sporocarps(10% of the cases). From a total of 60 fungal isolatestested (Table 1), 17 (nine species) formed ectomycorr-hizas with containerized P. pinea seedlings (Tables 2,3). The morphological characteristics of the ectomy-corrhizas formed are summarized in Table 3. The per-centages of ectomycorrhizas formed by P. pinea seed-lings varied among species and among isolates of thesame species (Table 2). Rhizopogon roseolus isolateswere the most infective, producing percentages of my-

corrhizal short roots of 6389%. Lactarius chrysorrheusand Pisolithus tinctorius isolates showed a variable in-fectivity, with percentages of mycorrhizal short roots of3976% and 2271%, respectively. This variabilityamong isolates of the same species was also observed inHebeloma crustuliniforme and Laccaria laccata (Table2). Other species in the genera Boletus, Cortinarius,Hygrophorus, Leccinum, Lepista, Tricholoma and Xe-rocomus failed to form ectomycorrhizas under the ex-perimental conditions used in this study.

Discussion

As with other synthesis methods, the non-axenic syn-thesis used here was done under relatively artificialconditions and the results must be interpreted cautious-ly when drawing conclusions about their natural occur-rence. Formation of ectomycorrhizas under synthesisconditions indicates that such a fungus-plant associa-tion is possible, but negative results are not conclusiveand at best suggest that such relationships seem im-probable (Molina and Palmer 1982; Brundrett et al.1996).

Fungal species belonging to the generaAmanita, Bo-letus, Cortinarius, Tricholoma andXerocomus failed toform ectomycorrhizas with containerized P. pinea seed-lings, although members of these fungal genera havebeen cited as ectomycorrhizal with other tree species(Trappe 1962). Similar results withBoletus, Tricholomaand Xerocomus species have been reported for othertree species (Kropp 1982; Kropp and Trappe 1982;Godboud and Fortin 1983; Danielson et al. 1984; Peraand Alvarez 1995; Parlade et al. 1996). These fungi maybe particularly prone to losing infectivity when grownin artificial media (Marx 1981) or the type of inoculum

used in these experiments may not have been optimalfor these fungi. All of these fungal genera belong to thelate-stage group of fungi that form symbioses with ma-ture trees instead of seedlings (Deacon and Fleming


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Table 1 Fungal isolates tested in synthesis of ectomycorrhizaswith containerized Pinus pinea seedlings (Aalb Abies alba, FsylFagus sylvatica, Phal Pinus halepensis, Pmen Pseudotsuga men-

ziesii, Pnig Pinus nigra, Ppin Pinus pinea, Ppter Pinus pinaster,Prad Pinus radiata, Psyl Pinus sylvestris, Qile Quercus ilex, QpubQuercus pubescens, Qsub Quercus suber)

Fungusa Isolate Potential Year Locationnumber associated host isolated

Amanita sp. 185 Ppter, Prad 1994 Bredac

Amanita citrina (Schaeff. ex) S. F. Gray 181 Ppin, Qile 1994 Cabrera de Marb

Amanita pantherina (D.C. ex Fr.) Kummer 207 Qsub, Ppin 1994 Forallac (Fitor)c234 Psyl 1995 St. Hilari Sacalmc

Amanita rubescens (Pers. ex Fr.) S. F. Gray 208 Qpub 1994 Forallac (Fitor)cBoletus sp. 203 Ppin, Qile 1994 Bredac

241 Qsub 1995 Llinars del Vallsb260 Qsub, Qile 1995 Forallac (Fitor)c267 Ppter 1995 Forallac (Fitor)c268 Ppter 1995 Forallac (Fitor)c

Boletus erythropus (Fr. ex Fr.) Pers. 292 Aalb 1995 Fogars de MontclsbCortinarius sp. 258 Fsyl 1995 Fogars de MontclsbHebeloma crustuliniforme (Bull. ex St. Amans) 265 Abies sp. 1995 Arbciesc

290 Abies sp. 1995 ArbciescHygrophorus russula (Schaeff. ex Fr.) Qul. 225 Ppter 1994 Fogars de MontclsbLaccaria laccata (Scop. ex Fr.) Berk. & Br. 127 Qile 1989 Arbciesc

226 Qsub, Ppin 1994 Forallac (Fitor)c

Lactarius chrysorrheus Fr. 231 Ppin 1994 St Celonib273 Ppin 1995 Forallac (Fitor)c275 Qsub 1995 Arbciesc201 Ppin, Qile 1994 Bredac262 Pnig 1995 Arbciesc

Lactarius deliciosus L. ex Fr. 178 Ppin 1994 Cabrera de Marb216 Ppin 1994 Forallac (Fitor)c274 Ppin 1995 Forallac (Fitor)c

Lactarius sanguifluus Paulet ex Fr. 261 Prad 1995 Arbciesc263 Ppter 1995 Forallac (Fitor)c

Leccinum sp. 278 Qsub, Qile 1995 Forallac (Fitor)cLepista nuda (Bull. ex Fr.) Cooke 209 Qsub 1994 Forallac (Fitor)cPisolithus tinctorius (Pers.) Coker & Couch. 93 Qsub 1986 Tossa de Marc

193 Qsub, Ppin 1994 Sta Coloma de Farnersc

202 Qsub 1994 Forallac (Fitor)

c

233 Qsub 1995 St Hilari Sacalmc249 Qile, Qsub 1995 Fogars de Montclsb

Rhizopogon luteolus Fr. & Nordh. 252 Pnig 1995 Fogars de MontclsbRhizopogon roseolus (Corda ex Sturm) Fr. 173 Ppin 1994 Orriusb

196 Ppter, Prad 1994 Bredac183 Ppin 1994 Bredac179 Ppter 1994 Mozoncillo (Segovia)294 Prad 1995 Bredac

Russula sp. 200 Ppin, Qub 1994 PalautorderacRussula foetens Pers. ex Fr. 269 Qsub, Qile 1995 Forallac (Fitor)cScleroderma sp. 281 Abies sp. 1995 ArbciescScleroderma verrucosum Bull. ex Pers. ss. Grv. 266 Abies sp. 1995 ArbciescSuillus sp. 164 Phal 1994 BredacSuillus bellinii (Inz.) Watling 174 Ppin 1994 Cabrilsb

240 Qsub 1995 Llinars del Vallsb

255 Ppin 1995 Forallac (Fitor)cSuillus granulatus (L. ex Fr.) O. Kuntze 232 Psyl 1995 St Hilari Sacalmc

277 Ppter 1995 Forallac (Fitor)cSuillus luteus (L. ex Fr.) S. F. Gray 251 Fsyl 1995 Forallac (Fitor)cTricholoma spp. 175 Ppin, Qile 1994 VallgorguinabXerocomus badius (Fr.) Khner ex Gilbert 167 Qsub 1994 Sta Coloma de Farnersc

271 Qsub, Qile 1995 Forallac (Fitor)cXerocomus ferrugineus Schaeffer 168 Qsub 1994 Sta Coloma de FarnerscXerocomus subtomentosus (L. ex Fr.) Qul. 176 Ppin, Qsub 1994 Vilalba Saserrab

177 Ppin, Qsub 1994 Palautorderac247 Qile, Qsub 1995 Fogars de Montclsb250 Qsub 1995 St Feliu de Buixalleuc279 Qsub 1995 Forallac (Fitor)c

a Voucher specimens of those fungal isolates that formed ectomycorrhizas with containerized P. pinea seedlings were deposited in theReal Jardn Botnico de Madrid Herbarium (Spain), others are being kept in the herbarium of the Department de Patologia Vegetal(IRTA)b Barcelonac Girona


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Table 2 Fungal isolates that formed ectomycorrhizas with containerized P. pinea seedlings (new syntheses with numbers) and ectomy-corrhizal fungi previously cited in the literature for this conifer ( not given)

Fungus Isolate number Mycorrhizaeor literature reference colonization

(%BSD)

Amanita sp. 185 56B20.2

Cenococcum geophilum Fr. Trappe (1962) Hebeloma crustuliniforme (Bull. ex St. Am.) Qul. 290 45B5.6265 13B6.8Branzanti et al. (1985) 87

Hebeloma sinapizans (Paul. ex Fr.) Gillet Branzanti and Zambonelli (1988) 90Laccaria laccata (Scop. ex Fr.) Bk. & Br. 127 24B13.2

Branzanti et al. (1985) 93Branzanti and Zambonelli (1988) 72

Lactarius chrysorrheus Fr. 275 71B8.1262 22B18.8

Lactarius deliciosus (L. ex Fr.) S. F. Gray 274 50B15.2Trappe (1962)

Lactarius hygrophoroides Berk & Curt. Barsali (1922) Paxillus involutus (Batsch. ex Fr.) Fr. Branzanti and Zambonelli (1988) 64Pisolithus tinctorius (Pers.) Cok, & Couch

(pP. arhizus (Scop:Pors.) Rausch.) 249 58B5.6193 39B14.893 76B13.0

Mousain et al. (1987; 1994) Rhizopogon roseolus (Corda ex Sturm) Fries 179 89B9.9

183 88B5.8196 63B24.9Mousain et al. (1987; 1994)

Scleroderma sp. 281 82B15.7Suillus collinitus (Fr.) O. Kuntze Mousain et al. (1987; 1994) Suillus bellinii (Inz.) Waltling 164 48B16.2

174 48B15.9240 41B11.6

Suillus granulatus (L. ex Fr.) O. Kuntze Takacs (1961) Branzanti and Zambonelli (1988) 58

Suillus luteus (L. ex Fr.) S. F. Gray Trappe (1962) Tuber albidum Pico Branzanti and Zambonelli (1984)

Pascualini et al. (1992) Tuber borchii Vitt. Barsali (1922)

Ceruti (1965)

Table 3 General morphological characteristics of ectomycorrhizas formed on P. pinea

Fungus Isolate Color of mantle Hartig neta Mycelial Rhizomorphs/growthb mycelial strandsc

Amanita sp.d 185 White cc c ccHebeloma crustuliniforme 290 White cc ccc P

265 White cc ccc PLaccaria laccata 127 Beige-violet cc c PLactarius chrysorrheusd 275 Beige-pale orange cc c P

262 Beige-pale orange cc c PLactarius deliciosus 274 Orange-green cc c PPisolithus tinctorius 249 Yellow ccc ccc ccc

193 Yellow ccc ccc ccc93 Yellow ccc ccc ccc

Rhizopogon roseolus 179 White-pink ccc ccc ccc183 White-pink ccc ccc ccc196 White-pink ccc ccc ccc

Scleroderma sp.d 281 White cc ccc ccSuillus belliniid 164 White-pink cc ccc cc

174 White-pink cc ccc cc240 White-pink cc ccc cc

a Hartig net development: c outer layer of cortex cells, cc first and second layers of cortex, ccc first to third layers of cortexb Mycelial growth extended from mantle: P undetectable,c sparse, cc abundant, ccc very abundantc Rhizomorphs or mycelial strands: P absent, c scarce, cc abundant, ccc very abundantd First report of this species together with P. pinea


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1992). Other fungal isolates belonging to the generaHygrophorus, Leccinum and Lepista did not form ecto-mycorrhizas with P. pinea. Some species of these gen-era are known to be saprophytic, while others havebeen reported more frequently in association with an-giosperms than with gymnosperms (Trappe 1962). Thenegative results do not exclude the possibility that P.

pinea could form ectomycorrhizas with these fungi un-der natural conditions but it precludes future evalua-tions of these fungi for seedling inoculation.

Most of the isolates that formed ectomycorrhizaswith containerized P. pinea seedlings belong to speciescommonly observed in association with stone pine inthe field: Lactarius spp., Pisolithus tinctorius, Rhizopo-

gon roseolus and Suillus spp. Of the 17 isolates thatformed ectomycorrhizas, nine infected over 50% of P.

pinea short roots. Such information may be useful inselecting aggressive fungal isolates for nursery inocula-tions with vegetative inocula. The colonization abilityof a fungus is a selection criterium for candidates innursery inoculation programs. It has been suggestedthat the colonization level of the root system is relatedto the fungal effect on outplanted seedlings (Marx1980; Ruehle et al. 1981; Trofymow 1990), although theminimal colonization level needed to ensure expressionof a potential beneficial effect is unknown for largenumbers of EM fungi. Among the 17 isolates whichformed ectomycorrhizas with P. pinea in our experi-ments, Laccaria laccata and Pisolithus tinctorius areknown to be broad-host-range, worldwide fungi (Moli-na 1982; Marx 1991). These, in addition to Hebelomacrustuliniforme, Rhizopogon roseolus and Suillus spp.have been cited previously for P. pinea (Table 2). Ourresults extend the number of EM fungi of P. pinea to

Amanita sp., Lactarius chrysorrheus, Scleroderma sp.and Suillus bellinii.

Rhizopogon, Scleroderma and Suillus spp. producelarge quantities of rhizomorphs, structures related tothe enhancement of water uptake by the plant (Dud-dridge et al. 1980; Cairney 1992). This characteristiccould be of interest in the selection of ectomycorrhizalfungi for P. pinea, which is usually grown under the dryconditions associated with a Mediterranean climate.

The difficulty in obtaining EM seedlings inoculatedwith mycelial inoculum of Rhizopogon spp. has beenreported previously (Molina 1980; Molina and Trappe1994). The results obtained in this present study indi-cate the possibility of inoculating P. pinea seedlingswith Rhizopogon mycelia at high inoculum rates, al-though, for practical application in nursery inocula-tions, more research is needed to determine the successof this type of inoculum when applied at lower rates.

The results obtained are the beginning of an ectomy-corrhizal selection process to obtain P. pinea seedlingsinoculated with ectomycorrhizal fungi and suitable to

withstand Mediterranean field conditions. Field experi-ments are currently in progress to determine the per-formance of P. pinea seedlings inoculated with most ofthe above-mentioned EM fungi (Parlad et al. 1997,

1998). At the same time, currently available moleculartechniques (Armstrong et al. 1989; Gardes et al. 1991)could become extraordinarily powerful tools for iden-tifying fungal species or fungal strains associated withtheir hosts in field situations, even on a single mycorr-hiza. This would help in assays of distribution, persis-tence and competitive ability of introduced fungi. Dataobtained on plant growth and survival, and on fungalpersistence, will enable the selection of the most effi-cient EM strains.

Acknowledgements This work was supported by the EuropeanCommission Contract No. AIR2- CT94-1149, and was part of thedoctoral thesis of the first author, granted by the Direccin Gen-eral de Investigacin Cientfica y Tcnica. Ministerio de Educa-cin y Ciencia, Spain. Mention of trade names or commercialproducts does not constitute endorsement or recommendation foruse.

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