Truffle Cultivation in Sweden: Results from Quercus Robur and Corylus Avellana Field Trials on the Island of Gotland

Scandinavian Journal of Forest Research, 2009; 24: 38Á54

ORIGINAL ARTICLE

Truffle cultivation in Sweden: Results from Quercus robur and Corylus avellana field trials on the island of Gotland

CHRISTINA WEDE´ N1, LINA PETTERSSON2 & ERIC DANELL3

1Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden, 2Kalmar- Kronoberg County Agricultural Society, Va¨xjo¨, Sweden, 3Museum of Evolution, Botany Section, Uppsala University, Uppsala, Sweden

Abstract The edible, ectomycorrhizal Burgundy truffle, Tuber aestivum Vitt., grows naturally on the islands of Gotland and O¨ land, Sweden. In 1999, 240 inoculated Quercus robur and Corylus avellana seedlings inoculated with French T. aestivum were planted in 10 experimental truffle orchards (truffie`res) on Gotland to investigate the possibility of truffle cultivation in Sweden. Truffle orchard management, mycorrhizal development and seedling growth were studied. Fourteen additional truffle orchards containing more than 2000 Q. robur were established during 2000 and 2001. In 2004, T. aestivum mycorrhizae were detected in all truffle orchards. In 2005, the first T. aestivum truffle was found, 6 years after planting. This is the first cultivated truffle in Scandinavia and despite the northerly location they were produced within a timescale comparable with France. Tuber aestivum mycorrhizae survived in soils which differed from naturally producing locations by having a sand content 95%, pH 56.4 and calcium content 50.1%. In a second series of experiments Swedish Q. robur, C. avellana and Carpinus betulus seedlings were inoculated with Swedish T. aestivum. Eleven months after inoculation T. aestivum mycorrhizae were found in 0Á78% of the seedlings, depending on species, inoculation treatment and substrate. Because of the continuing decline of oaks in Sweden and associated fauna and flora, truffle cultivation may favour oak forest restoration programmes as well as making a direct contribution to rural economies.

Keywords: Carpinus betulus, Corylus avellana, mycorrhiza, Quercus robur, trufﬂe cultivation, Tuber aestivum, Tuber uncinatum.

Introduction T. aestivum is used throughout this article. Owing to its hypogeous nature, truffle spores are not spread by Truffles, the macrofungi forming underground (hy- wind or water, but instead at spore maturity the pogeous) fruit bodies, belong to diverse groups of truffle fruit body emits volatile sulphurous hydro- both ascomycetes (spore sac fungi) and basidiomy- carbons that attract potential spore vectors such as cetes. Most truffle species live in ectomycorrhizal rodents or deer (Bellina-Agostinone et al., 1987; association with various trees and shrubs. Ascomy- Trappe, 1988; Trappe & Castellano, 1991). These cete truffle species are commonly referred to as ‘‘true eat the truffle and spread concentrated packets of truffles’’ and basidiomycete truffles as ‘‘false truf- spores in the faeces. The volatile substances are also fles’’. Several members of the true truffle genus Tuber the reason why some truffle species are highly are highly appreciated as delicacies. One of these appreciated as delicacies that command high prices species is the black Burgundy truffle (Tuber aestivum on the world market (Olivier et al., 1996). The Vitt. syn. T. uncinatum Chat.), which in Scandinavia market demand has also led to the development of is known from the islands of Gotland and O¨ land commercial truffle cultivation on inoculated tree (Sweden) and from Denmark. Since T. uncinatum is seedlings over the past two centuries (Chevalier & a synonym for T. aestivum (Paolocci et al., 2004; Frochot, 1997a). The Burgundy truffle forms ecto- Wede´n, 2004; Wede´n et al., 2005), the older name mycorrhizae with, for example, oak (Quercus spp.),

Correspondence: C. Wede´n, Division of Pharmacognosy, Department of Medicinal Chemistry, BMC, Box 574, Uppsala University, SE-751 23 Uppsala, Sweden. E-mail: [email protected]

(Received 27 May 2008; accepted 17 October 2008) ISSN 0282-7581 print/ISSN 1651-1891 online # 2009 Taylor & Francis DOI: 10.1080/02827580802562056 Truffle cultivation in Sweden 39 beech (Fagus sylvatica), hornbeam (Carpinus betulus), augment truffle production over time (Chevalier & linden (Tilia spp.) and hazel (Corylus spp.) (Cheva- Grente, 1979). This form of truffle cultivation is still lier & Frochot, 1997a). The natural geographical practised today, and is referred to as the Talon distribution of T. aestivum ranges from North Africa method. The mutualistic relationship between fun- to Sweden and from Ireland to Russia (Chevalier & gus and tree was postulated by Frank (1885), who Frochot, 1997a; Wedeń & Danell, 1998). Tub er discovered the association between T. aestivum aestivum, found in 1997 at a new site on the island mycelia and fine roots, naming it mycorrhiza (Gr. of Gotland, had large fruit bodies with mature spores mykosfungus and rhizaroot). With this new suggesting good adaptation to soil and climatic understanding of the T. aestivum life cycle, and that conditions (Wedeń & Danell, 1998). Before this, of many other fungi, Malençon (1938) introduced T. aestivum had been reported only three times from the idea of planting seedlings already harbouring Sweden (Gotland) and was considered endangered truffle mycorrhizae. When wild truffle harvests in and extremely rare (Larsson, 1997). In 1998, the France, Italy and Spain progressively decreased landowner of the newly discovered site found 5 kg of during the first part of the twentieth century, the T. aestivum within an area of 50 m2. Cultivation of need for reliable truffle cultivation techniques be- T. aestivum has been developed in France and Italy came obvious. In the late 1960s and 1970s, success- (Chevalier & Frochot, 1987, 1989, 1997b, 2002; ful production of Tuber mycorrhizae in greenhouse Chevalier & Dupre´, 1988; Giovanetti et al., 1994; experiments was demonstrated (Fontana, 1967; Belloli et al., 2001). Because of an ongoing decline Palenzona, 1969; Fontana & Bonfante-Fasolo, of oaks in Sweden since the 1700s (Sta˚a˚l, 1994; 1971; Grente et al., 1972; Chevalier & Desmas, L. Kardell, Swedish University of Agricultural 1975; Delmas & Poitou, 1979). In 1976, the first Sciences, personal communication), associated truffles (T. melanosporum) were produced in a truffle fauna and flora are becoming rare, making econom- orchard (truffie`re) of inoculated seedlings in Italy, ical incentives for planting oak an important goal per 6 years after planting (G. Chevalier, French National se. A 5-year research project was therefore estab- Institute for Agricultural Research, personal com- lished in 1999 to study natural populations of munication). In 1977 the first truffles (T. melanos- T. aestivum on Gotland, seedling inoculation techni- porum) from inoculated seedlings were produced in ques and truffle cultivation in Sweden with the France, 3.5 years after planting (Chevalier & Grente, overall aim of causing socioeconomic effects, parti- 1979). Seedling inoculation has also made it possible cularly on the rural island of Gotland (Figure 1). to establish productive truffle orchards outside the Truffles have historically been collected in the natural geographical distribution of these truffles, wild, but Joseph Talon discovered in the early 1800s e.g. in New Zealand and North America (Hall et al., that planting acorns in natural truffle regions could 1998a; Hall & Wang, 2002). Currently, commercial mycorrhizal truffle seed- 10°E 15°E 20°E 25°E lings are produced using mainly T. melanosporum (black Pe´rigord truffle) and T. aestivum (black Burgundy truffle) (Chevalier & Frochot, 1987, 1989; Hall et al., 1998b). Because of the commercial value of successful inoculation techniques, large truffle seedling producers guard their methods well (Hall et al., 2003, 2007). However, successful Sweden inoculations of numerous host species with various 60°N 60°N Tuber species have been reported (Table I). Table II lists the reported mycorrhiza synthesis of T. aestivum Gotland with different plant species and inoculation methods. Roots of an ectomycorrhizae-forming tree or plant can be inoculated with an ectomycorrhizal fungus by contact with germinating spores, mycelia or mycorrhizae. Pieces of a root system, or an entire plant 55°N 55°N with mycorrhizae, could be potted together with the seedlings to be inoculated (Chevalier & Grente, 1973). Mycorrhizae may develop on the new seed- 10°E 15°E 20°E 25°E lings within 2Á3 months and the method is applic- Figure 1. Map of southern Scandinavia showing the position of able to many Tuber species (Giovanetti et al., 1994). the Swedish island of Gotland, off the Swedish east coast. Mycelial inoculation requires truffle mycelium ob- (Illustration: Anders Larsson.) tained in pure culture on artificial substrate. Such 40 C. Wedeń et al.

Table I. Reported mycorrhiza synthesis of Tuber species.

Tuber sp. References

Tuber aestivum Palenzona (1969), Chevalier et al. (1973, 1975), Chevalier & Desmas (1975), Giovannetti & Fontana (1982), Zambonelli & Branzanti (1984), Zambonelli & Govi (1988), Zambonelli et al. (1993), 1995), Donnini et al. (2003), Pruett et al. (2008) Tuber borchii Fassi & Fontana (1967), Fontana (1967), Fontana & Palenzona (1969), Fontana & Bonfante- Fasolo (1971), Giovannetti & Fontana (1982), Zambonelli & Branzanti (1984), 1989), Zambonelli et al. (1993), 1995, 2002), Granetti et al. (1995), Sisti et al. (1998), Giomaro et al. (2000) Tuber brumale Palenzona (1969), Palenzona et al. (1972), Chevalier (1973), Chevalier & Desmas (1975), Giovannetti & Fontana (1982), Zambonelli et al. (1993, 1995), Giomaro et al. (2002), Donnini et al. (2003) Tuber macrosporum Giovannetti & Fontana (1980Á81, 1982), Zambonelli et al. (1993) Tuber maculatum Fontana (1967), Fassi & Fontana (1969), Parlade´ et al. (1996), Zambonelli et al. (1999) Tuber melanosporum Palenzona (1969), Grente et al. (1972), Palenzona et al. (1972), Chevalier et al. (1973), Chevalier & Desmas (1975, 1977a, b), Chevalier & Grente (1979), Delmas & Poitou (1979), Fontana & Giovannetti (1978Á79), Chevalier (1984), Guinberteau et al. (1988), Boutekrabt et al. (1990), Zambonelli et al. (1993, 1995), Rauscher et al. (1995), Fischer & Colinas (1996), Mamoun & Olivier (1996, 1997), Donnini et al. (1997), 2003), Pinkas et al. (2000), Wenkart et al. (2001), Kagan-Zur et al. (2002), Gong et al. (2003) Tuber mesentericum Chevalier et al. (1973), Giovannetti & Fontana (1982), Zambonelli et al. (1993, 1995), Rauscher et al. (1995) Tuber oligospermum Bencivenga et al. (1997) Tuber rufum Palenzona et al. (1972), Giovannetti & Fontana (1982), Rauscher et al. (1995) inoculation is faster than spore inoculation, but not tion). The risk of inoculating plants with pure efficient for large numbers of seedlings owing to the mycelial cultures is that the fungus may have a slow growth of Tuber mycelia on artificial substrate narrow genetic base and perhaps be adapted only to (Boutekrabt et al., 1990; Iotti et al., 2002). Pure certain narrowly defined ecological conditions (Hall cultures of a Tuber species were first obtained by et al., 1998a; Hall & Wang, 1998). Limited genetic Fontana (1968). The report of T. melanosporum in diversity may also limit the potential for sexual pure culture by Matruchot (1903) was later proven reproduction and fruit body formation (Paolocci to be mistaken (G. Chevalier, personal communica- et al., 2006; Rubini et al., 2007). At present it is

Table II. Reported mycorrhiza synthesis of Tuber aestivum (syn. T. uncinatum) with different plant species.

Plant species Inoculation method References

Castanea sativa Spore Chevalier et al. (1973), Zambonelli & Govi (1988) Cistus incanus ssp. incanus Spore Giovannetti & Fontana (1982) Cistus spp. Spore Chevalier et al. (1975) Corylus avellana Spore Palenzona (1969), Chevalier et al. (1973), Zambonelli & Branzanti (1984), Zambonelli & Govi (1988) Fagus sylvatica Spore Chevalier et al. (1973), Zambonelli & Govi (1988) Ostrya carpinifolia Spore Zambonelli & Govi (1988), Donnini et al. (2003) Pinus halepensis Spore Chevalier et al. (1973) Pinus nigra Spore Chevalier et al. (1973), Zambonelli & Govi (1988) Pinus pinea Spore Zambonelli et al. (1995) Pinus strobus Spore Chevalier et al. (1973) Pinus sylvestris Spore Chevalier et al. (1973), Zambonelli & Govi (1988) Mycelium Chevalier & Desmas (1975) Quercus cerris Spore Zambonelli & Govi (1988) Quercus coccifera Spore Chevalier et al. (1973) Quercus ilex Spore Chevalier et al. (1973) Quercus peduculata/ Quercus robur Spore Chevalier et al. (1973), Zambonelli & Govi (1988), Pruett et al. (2007) Quercus pubescens Spore Chevalier et al. (1973), Zambonelli & Govi (1988), Zambonelli et al. (1993), Donnini et al. (2003) Quercus sessiliflora Spore Chevalier et al. (1973) Tilia cordata Spore Chevalier et al. (1973)

Note: Inoculation was performed with either spores from fruit bodies of T. aestivum, or mycelia in pure culture, derived from mycelia from the gleba of T. aestivum fruit bodies. Truffle cultivation in Sweden 41

‘‘safer’’ to use spores as inoculum, although mosaics seedling, either by covering the soil with straw of genotypes may slow root system colonization. (exchanged for hay in truffle orchard 2A), chipped With the use of spores as inoculum there is also a conifer bark or white plastic weave (Supermat, potential risk of contamination of spores from other Tra¨dga˚rdsteknik, A¨ ngelholm), or by manual weeding fungi, unlike when a pure mycelial culture is used. (Table III). Between rows, the soil was ploughed, Inoculation with spores is still the most common harrowed or weeded manually, or covered by reg- method for commercial truffle seedling production, ularly mown grass. In all 10 truffle orchards irrigation because spores are a readily available and relatively was recommended in periods of summer drought, inexpensive source of inoculum (Parlade´ et al., especially during the first months after planting. 1996). Spore inoculation is performed under semi- The 10 experimental truffle orchards were estab- sterile conditions, using spore suspensions prepared lished to study mycorrhizal survival, seedling devel- from fresh, frozen or dried fruit bodies (Parladeét opment and different management schemes during a al., 1996). Techniques for successful inoculation maximum number of seasons within the 5-year differ among Tuber species and host species. project. Because a large percentage (15%) of the The aim of this study was to investigate: (1) the farmers on Gotland practise organic farming (M. possibility of inoculation of seedlings of three differ- Wallman, Swedish Board of Agriculture, personal ent tree species which have shown good indications communication), weed control was tested without for T. aestivum cultivation and which occur naturally using herbicides. Root samples were taken yearly in in Sweden, and (2) the possibility of truffle cultiva- all truffle orchards to follow the development of tion in Sweden with respect to survival and propaga- T. aestivum, and to be able to take measures in the tion of T. aestivum mycorrhiza, fruit body (truffle) case of failure to detect T. aestivum mycorrhiza. Sites production and truffle orchard management. as diverse as possible were selected, covering different geographical locations on Gotland, to enable a preliminary assessment of suitable soil and location Materials and methods types. All soils were well drained and had not been Ten truffle orchards were established on the island of populated by trees for at least 20 years, to lower Gotland in 1999. The occurrence of T. aestivum competition by other mycorrhizal fungi (Chevalier & mycorrhiza was followed during 2000Á2006 and fruit Frochot, 1997a; Smith & Read, 1997; Hall et al., body production studied by searching the orchards 2007). All experimental seedlings were financed by with a trained truffle dog during the seasons 2004Á the truffle project, but owned and managed by the 2007. Data for the study were added by the establish- landowners. As a consequence all of the truffle ment of another 14 commercial orchards during orchards contained seedlings from the same batch 2000Á2001, and these orchards were followed during and were managed the same, but had differing soils 2001Á2004. In a separate inoculation experiment, and managers. Swedish tree seedlings of Quercus robur, Corylus avellana and Carpinus betulus were inoculated with Establishment of commercial truffle orchards spores from Swedish T. aestivum fruit bodies to study the effects of pot substrate, tree species and time on As a consequence of the truffle inventories (Wedeń & T. aestivum mycorrhiza formation. Danell, 1998; Wedeń et al., 2001, 2004a,b), interest in commercial production rose. Another 3000 Q. robur seedlings were planted on Gotland by Establishment of experimental truffle orchards individual landowners in 2000 and 2001. These In 1999, 10 T. aestivum truffle orchards were estab- seedlings were produced by Robin Pe´pinie`res, lished on Gotland, a Swedish island where T. aestivum France, using T. aestivum inoculum from Gotland. grows naturally (Wedeń et al., 2004a). Each truffle Swedish Q. robur acorns were unavailable so they orchard (Table III) consisted of 12 Q. robur seedlings were sourced from Denmark instead. One truffle purchased from Robin Pe´pinie`res and 12 C. avellana orchard was established after removing stems and seedlings purchased from Agri-Truffe. The seedlings roots of the existing trees (primarily Populus sp., and the T. aestivum inoculum used to produce the some Betula sp. and Pinus sylvestris), 1 month before mycorrhiza originated from northern France. The planting. The remaining 13 truffle orchards were 1.5-year-old seedlings were delivered in June 1999. planted on old agricultural or meadowland without All but two to five leaves were removed without recent tree growth. Seedlings were weeded manually, harming the new leaf bud, to prevent dehydration by mulching with straw or cardboard discs (40 cm after planting. Seedlings were planted 3Á4 m apart diameter), or not at all (Table IV). Seedlings were along two or three rows 4Á6 m apart. Weed control protected against rabbits, hares and grazing animals was carried out within at least 1 m around each by caging seedlings by wire mesh, by fencing the 42 C. Wedeń et al.

Table III. Investigation of 10 experimental trufﬂe orchards established in June 1999 on Gotland, Sweden, with 120 Quercus robur (Robin Pe´pinie`res, France) and 120 Corylus avellana (Agri-truffe, France) seedlings inoculated with T. aestivum (syn. T. uncinatum).

August 1999: 2004: No. of Seedling Seedling Site No. of seedlings seedlings protection Weed control Main cause of death height (m)

1A 5 (21%) 0 None None Competition from weeds, Á 2A 24 (100%) 23 (96%) Net cages 1/3 plastic, 1/3 bark and 1/3 straw Unknown 1.5 3A 23 (96%) 22 (92%) Net cages Manual weeding Unknown 1.5 4A 21 (88%) 19 (79%) None 1/3 plastic, 1/3 bark and 1/3 straw Unknown 1.5 5A 22 (92%) 20 (83%) None 1/3 plastic,a 1/3 bark and 1/3 straw Broken by plastic/wind 1.5 6A 21 (88%) 20 (83%) Net cages 1/3 plastic,a 1/3 barkb and 1/3 straw Competition from weeds B0.5 7A 23 (96%) 21 (88%) None Manual weeding/mown grass Eaten by hares/rabbits 0.5Á1.5 8A 24 (100%) 23 (96%) None 1/3 plastic, 1/3 bark and 1/3 none Competition from weeds 1.5 9A 24 (100%) 22 (92%) Net cages 1/3 plastic, 1/3 bark and 1/3 straw Unknown 1.5 10A 5 (21%) 0 None None Competition from weeds Á 192 (80%) (95%)c 170 (71%) (89%)c

Note: seedlings and truffle inoculum originated from northern France. Each of the 10 truffle orchards consisted of 12 Q. robur and 12 C. avellana seedlings. aIn orchards 5A and 6A, the plastic weave was removed during the first year. bChipped bark was applied in B 20 cm thick layer, ineffective against weeds within 2 years, as was also the case with the straw. Some manual weeding and grass mowing has been undertaken since. cPercentage surviving seedlings if the orchards 1A and 10A, where weed control was not executed, are excluded. August 1999: No. of seedlingsnumber of surviving seedlings in August 1999; 2004: No. of seedlingsnumber of surviving seedlings in 2004; Seedling protectionprotection against rodents, deer or grazing animals; Weed controlweed control 1 m around each seedling; Main cause of deathmain cause of seedling death; Seedling heightheight of majority of seedlings in 2004. entire truffle orchard, or not at all (Table IV). The Since quantitative sampling may have destroyed the area between the seedling rows, i.e. beyond 1 m from mycorrhizal tree seedlings in the truffle orchards, the the seedlings, was ploughed, harrowed, cultivated, decision was made simply to confirm whether grazed or covered with grass (mown or not). T. aestivum was still present in the truffle orchard. Five root samples were collected from five different seedlings in each truffle orchard. Seedlings were Root sampling and monitoring of all truffle orchards selected arbitrarily to represent a diagonal of the To assess whether T. aestivum was still present in the truffle orchard. Different seedlings were sampled truffle orchards, root samples were taken yearly every year. A soil bore (22 mm diameter) was used to between August and October, except for 2004 collect the samples from 5Á25 cm depth. When a when samples were collected at the end of May. seedling root was found in the soil core, it was rinsed

Table IV. Investigation of 14 Swedish trufﬂe orchards comprising in total 2149 Quercus robur (Danish acorn origin) inoculated with Tuber aestivum (origin Gotland) by Robin Pe´pinie`res, France.

No. of Site seedlings Planting date Seedling protection Weed control Seedling height (m)

11B 250 Oct. 2000 Net cages Some manual weeding. Cardboard discs 2002 0.5Á1.5 12B 200 Oct. 2000 Net cages Some manual weeding. Cardboard discs 2002 0.5Á1.5 13B 24 Oct. 2000 Net cages. Plastic spiral net 2004 None. Cardboard discs 2002 0.5Á1.5 14B 106 Oct. 2000 Net cages Manual weeding. Cardboard discs 2002 1.5 15B 102 Oct. 2000 None Manual weeding. Cardboard discs 2002 1.5 16B 150 Oct. 2000 Net cages Manual weeding. Cardboard discs 2002 0.5Á1.5 17B 560 Dec. 2001 Net fencing None. Cardboard discs 2002 B0.5 18B 125 June 2001 Net fencing Straw 30 cm packed 0.5Á1.5 19B 154 May 2001 None None B0.5 20B 130 Oct. 2000 Net fencing None. Cardboard discs 2002 B0.5 21B 30 Oct. 2000 Net cages None B0.5 22B 18 Oct. 2000 Net fencing None. Cardboard discs 2002 B0.5 23B 228 Oct. 2000 Net cages None/mown grass. Cardboard discs 2002 0.5Á1.5 24B 72 Oct. 2000 Net cages None. Cardboard discs 2002 0.5Á1.5 Total 2149

Note: only truffle orchards where more than 10 seedlings had survived the first year were followed in this study. No. of seedlingsnumber of planted seedlings; Seedling protectionprotection against rodents, deer or grazing animals; Weed control weed control 1 m around each seedling; Seedling heightheight of majority of seedlings in 2004. Truffle cultivation in Sweden 43 in water, put in a plastic bag and examined the same ber 2005, 29 NovemberÁ1 December 2006 and 14Á day or put in a tube with 70% ethanol for later 16 November 2007. Wede´n trained her own dog for examination. In truffle orchards where seedling this purpose. height had exceeded 1 m in 2003 and 2004, a main root was followed from the stem to find lateral Soil analyses roots for sampling. This was done since it was hard to find fine roots with the soil bore, owing to the Soil analyses were conducted in all truffle orchards expansion of the root system as the seedlings grew before or after planting. The soil samples were larger. Weed management, survival and vitality of analysed according to Wede´n et al. (2004a) by the seedlings, and seedling parasites were recorded. INRA soil laboratories (Arras, France).

Identification of mycorrhiza Seedling inoculation experiment The ectomycorrhiza formed by T. aestivum (Figure 2) To save time, 271 1Á2-year-old potted seedlings can be identified as ochreous or chestnut brown, produced by VegTech (Sweden) were inoculated: swollen fine roots, with the tip embedded in radiat- 105 Q. robur (provenance Uppland, Sweden), 66 ing twisted hyphae or cystidae (Chevalier & Frochot, C. avellana (provenance southern Sweden) and 100 1997a). Identification of mycorrhiza was conducted C. betulus (provenance Kronoberg, Sweden). The 3 under a dissecting microscope. When at least one seedlings were grown in trays of 93 cm pots. The root sample from a truffle orchard contained at least Q. robur, C. betulus and C. avellana seedlings were 1, one typical fresh T. aestivum mycorrhizal tip, 1 and 2 years old at the time of inoculation. All T. aestivum was recorded as still being present in seedlings were dormant when they were moved into the truffle orchard. A classification of the results the greenhouse (208C, 16 h light) in November from the mycorrhizal sampling over 5 years was 2000, 1 month before inoculation. Their roots were done, with emphasis on the sampling in 2004 (Table generally undamaged and healthy, although very Va, b). In 2000, five mycorrhizal samples from the dense. Some Laccaria sp. mycorrhiza was found on the root systems. 10 experimental truffle orchards on Gotland, identi- 1 fied as T. aestivum mycorrhizae in different stages of The number of spores g (fresh weight) fruit body was estimated for two fruit bodies (100% development, were selected and sent to G. Chevalier mature spores) of T. aestivum from Gotland. A piece (INRA Clermont-Ferrand, France) and A. Zambo- of the gleba (the spore-producing interior of the nelli (Universita` di Bologna, Italy) for confirmation truffle) was homogenized in tap water, and the of identity. number of spores ml1 in three aliquots was counted microscopically using a haemocytometer. Fruit body production The overall mean number of spores gÁ1 fruit body was calculated to be 4.89 106 (s 1.85 106), a The truffle orchards established in 1999 (Table III) figure in agreement with the findings of Parlade´ et al. were screened for truffle fruit bodies with trained (1996). Fruit bodies of T. aestivum from Gotland truffle dogs on 9Á13 October 2004, 28Á30 Novem- were stored at Á208C for 2 months before inoculation. Spore suspensions containing 5105,5106 and 5107 spores per seedling were prepared from fruit bodies homogenized in a blender with tap water. To test whether mechanical breakdown of asci influenced the degree of seedling colonization, homogenized gleba was thereafter also ground in a mortar for approximately 10 min before mixing with water, according to Fischer and Colinas (1996). Two different soil substrates were used. Sieved (5 mm) field soil, pH 7.6, collected adjacent to a natural truffle site on Gotland (site E-T8 in Wede´n et al., 2004a), was mixed with coarse perlite (Nordisk Perlite, Denmark) (4:1 v/v) and is referred to below as modified truffle orchard soil (MTOS). Peat (Hasselfors Garden, Sphagnum special, Super- Figure 2. Tuber aestivum mycorrhizal root tip with radiating fine) and coarse perlite was mixed (1:1 v/v), referred mycelia. (Photograph: Lina Pettersson.) to below as peat:perlite. Crushed limestone 44

Table V. Soil composition at (a) eight Tuber aestivum orchards established in 1999 and (b) 14 Tuber aestivum orchards established in 2000Á2001 on the island of Gotland, Sweden. Wede C.

Site ´ tal. et n (a) 1999 2Aa 3Aa 4Aa 5Aa 6Aa 7Aa 8A 9A Natural range

% Clay 22.4 12.2 17.8 24.9 17.5 17.5 12.6 7.9 10.4Á32.6 % Fine silt 25.0 11.5 12.0 17.4 12.7 15.0 9.7 4.9 9.8Á64.7b % Coarse silt 15.0 3.5 8.2 8.6 7.4 11.2 5.3 2.6 % Fine sand 2.5 15.3 47.6 18.4 24.4 19.7 24.5 19.1 12.9Á79.8c % Coarse sand 35.1 57.5 14.4 30.7 38.0 36.6 47.9 65.5 Soil type Si SiSa SiSa SiC-Sa SiSa SiSa SiSa SaSi Water pH 8.01 7.98 7.85 8.18 7.94 7.83 7.3 7.3 6.8Á7.9 % CaCO3 22.3 2.380 0.691 4.170 5.49 5.68 0.400 0.200 0.1Á10.5 %P2O5 0.016 0.018 0.012 0.075 0.025 0.045 0.032 0.024 0.002Á0.120 % Ca 0.689 0.581 0.409 0.944 0.623 0.684 0.245 0.198 0.36Á1.07 % Mg 0.026 0.005 0.006 0.015 0.006 0.010 0.005 0.004 0.009Á0.045 % K 0.022 0.008 0.007 0.029 0.019 0.023 0.013 0.012 0.008Á0.063 Ca/Mg 26.40 113.9 71.75 62.93 100.5 65.80 52.13 46.05 12.4Á67.7 K/Mg 0.85 1.60 1.23 1.91 3.05 2.16 2.68 2.79 0.3Á4.0 % Organic C 3.46 3.13 1.96 3.38 2.74 4.9 1.449 2.032 3.5Á12.3 % Organic matter 5.99 5.42 3.39 5.85 4.73 8.48 2.49 3.500 6.0Á21.2 % Total N 0.361 0.230 0.170 0.298 0.239 0.461 0.141 0.180 0.3Á1.1 C/N 9.59 13.6 11.5 11.3 11.4 10.6 10.28 11.29 9.7Á18.2 Tuber aestivum Site

(b) 2000Á01 11Ba 12B 13Ba 14Ba 15Ba 16Ba 17Ba 18B 19Ba 20B 21B 22B 23B 24Ba

% Clay 3.4 20.2 5.3 6.0 4.4 13.9 3.3 30.5 19.5 21.0 17.0 18.8 13.5 7.2 % Fine silt 0.8 26.0 2.3 2.9 2.2 11.9 0.8 21.5 15.6 16.6 13.3 15.4 8.6 3.7 % Coarse silt 1.2 13.2 1.5 1.9 2.0 10.5 1.7 5.5 8.8 8.2 6.4 8.3 2.3 1.9 % Fine sand 80.1 21.0 79.1 74.2 77.2 32.6 40.7 29.0 23.3 20.1 13.2 19.8 18.5 52.4 % Coarse sand 14.5 19.6 11.8 15.0 14.2 31.1 53.5 13.5 32.8 34.1 50.1 37.7 57.1 34.8 Soil type Sa Si Sa Sa Sa SiSa Sa SiC SiSa SiC-Sa SiSa SiSa SiSa SaSi Water pH 7.11 8.2 6.36 7.2 6.50 8.00 6.41 7.8 7.59 7.40 8.00 8.00 8.00 7.49 % CaCO3 0.122 27.50 B0.1 B0.1 B0.1 2.060 0.100 1.900 0.327 0.600 12.90 16.6 5.500 0.143 %P2O5 0.012 0.013 0.003 0.006 0.021 0.014 0.007 0.009 0.036 0.003 0.008 0.014 0.004 0.002 %Ca 0.155 0.893 0.255 0.320 0.113 0.438 0.188 0.724 0.279 0.642 0.941 0.818 0.724 0.266 %Mg 0.004 0.013 0.014 0.024 0.004 0.004 0.005 0.019 0.008 0.008 0.008 0.013 0.006 0.004 % K 0.006 0.006 0.003 0.009 0.003 0.006 0.003 0.024 0.013 0.008 0.007 0.012 0.004 0.003 Ca/Mg 40.79 70.87 18.21 13.50 30.54 125.1 39.17 37.71 37.2 75.53 116.2 62.44 113.1 63.33 K/Mg 1.59 0.46 0.24 0.38 0.92 1.66 0.53 1.22 1.79 0.96 0.89 0.92 0.56 0.60 % Organic C 1.92 4.937 4.27 5.07 1.57 2.11 2.96 6.274 2.29 4.824 5.952 5.065 3.603 2.6 Table V (Continued) Site

(b) 2000Á01 11Ba 12B 13Ba 14Ba 15Ba 16Ba 17Ba 18B 19Ba 20B 21B 22B 23B 24Ba

% Organic matter 3.31 8.49 7.39 8.77 2.71 3.65 5.13 10.79 3.97 8.30 10.24 8.71 6.20 4.49 % Total N 0.142 0.532 0.29 0.335 0.145 0.168 0.225 0.646 0.171 0.345 0.496 0.526 0.335 0.213 C/N 13.5 9.28 14.7 15.1 10.8 12.6 13.2 9.71 13.4 13.98 12.00 9.63 10.76 12.2 Tuber aestivum

Note: atotal nitrogen content was measured in these soil samples, instead of organic nitrogen content; bsum of fine and coarse silt; csum of coarse and fine sand. Natural rangerange of soil parameters from soil analyses of naturally producing T. aestivum sites on Gotland. Bold numbers indicate values falling below, and italic numbers indicate values above the natural T. aestivum range on Gotland (Table 1 in Wede´n et al., 2004a): soil texture values falling below or exceeding the range with5%; pH0.2 outside the natural range; all other parameters less than half of lowest value or more than twice the highest value of the natural range. Clay (B 2 mm), fine silt (2Á20 mm), coarse silt (20Á50 mm), fine sand (50Á200 mm) and coarse sand (200Á2000 mm). Soil type: Cclayey; Sisilty; Sasandy. CaCO3 calcium carbonate; P2O5 phosphate; Cacalcium; Mgmagnesium; Kpotassium; Ccarbon; Nnitrogen. Tuber aestivumclassification of T. aestivum mycorrhiza vitality as measured by the probing method: presence of one or few T. aestivum mycorrhizae in one to three out of five samples, or indicating a receding trend since establishment; presence of numerous T. aestivum mycorrhizae in two to five out of five samples; presence of clusters of T. aestivum mycorrhizae with vigorous cystidiae in two to five out of five samples. rfl utvto nSweden in cultivation Truffle 45 46 C. Wede´n et al.

Table VI. Number of seedlings that had formed Tuber aestivum mycorrhiza after 4.5 months and after 11 months from inoculation.

Quercus robur No. of spores/pot; time

Substrate; inoculum 5105; 4.5 months 5105; 11 months 5106; 4.5 months 5106; 11 months

Peat:perlite 0% (0 of 5) 0% (0 of 6a) 0% (0 of 5) 33% (5 of 15) MTOS 0% (0 of 5) 40% (6 of 15) 0% (0 of 5) 67% (10 of 15) MTOSbroken asci 20% (2 of 10) 53% (8 of 15) 20% (2 of 10) 53% (8 of 15)

Carpinus betulus No. of spores/pot; time

Substrate; inoculum 5105; 11 months 5106; 11 months 5107; 11 months

Peat:perlite 0% (0 of 10) 20% (2 of 10) 50% (5 of 10) MTOS 20% (2 of 10) 10% (1 of 10) 78% (7 of 9b) MTOSdipped roots 50% (5 of 10) 20% (2 of 10) 50% (4 of 8c)

Corylus avellana No. of spores/pot; time

Substrate; inoculum 5105; 11 months 5106; 11 months 5107; 11 months

Peat:perlite 0% (0 of 9b) 0% (0 of 10) 11% (1 of 9b) MTOS 20% (2 of 10) 38% (3 of 8c) 44% (4 of 9b)

Note: inoculation by potting in substrates containing 5105,5106 or 5107 T. aestivum spores/seedling. Timetime from inoculation; MTOSmodified truffle orchard soil, i.e. unsterilized soil taken from a field adjacent to a naturally producing T. aestivum site on Gotland (site E-T8, Table 1 in Wede´n et al., 2004a). Numbers in parenthesesnumber of seedlings with T. aestivum mycorrhiza out of total number of checked seedlings. Quercus robur: Broken asciasci broken to liberate the ascospores prior to inoculation. Carpinus betulus: Dipped rootsseedling root system dipped in a slurry of T. aestivum spores and water before potting in inoculated MTOS. Corylus avellana: aremaining nine seedlings in batch dead; bremaining one seedling in batch dead; cremaining two seedlings in batch dead.

(Nordkalk, Tra¨dga˚rdsma¨starens kalk, CaO 50%) shoot development monthly during the first was added (1.8 g dmÁ3) to raise the pH of the 4 months. peat:perlite to 7, to confer a competitive on the Seedlings were checked for presence of T. aestivum T. aestivum mycelia relative to any pre-existing mycorrhizae 4.5 and 11 months following inocula- mycorrhiza. The original pH in the peat:perlite tion. For each treatment, five to 10 seedlings were mixture was 6.3Á6.5. examined. First, one-quarter of the root system was All seedlings were moved into a greenhouse cut out (sliced vertically along two radii) and (208C, 16 h light) 1 month before inoculation on examined under a dissecting microscope. If no T. 1 December 2000, while still dormant. Three differ- aestivum mycorrhizae were found, another quarter ent inoculation treatments were tested (Table VI): was examined. Seedlings were then put back in their (1) potting in inoculated substrate containing spore pots, adding peat:perlite to fill the empty space. If no suspensions of unbroken asci (Q. robur, C. avellana T. aestivum mycorrhizae were found on the first five and C. betulus); (2) potting in inoculated substrate seedlings examined in one treatment, the remaining containing spore suspensions of mechanically broken plants were left for further development. If truffle asci (Q. robur); and (3) immersing the root system in mycorrhizae were found in at least one of the first a spore suspension before potting in inoculated five seedlings, five more were examined. After substrate containing unbroken asci (C. betulus). 4.5 months, all Q. robur treatments were examined. Spore suspensions were first mixed with perlite, Only five of the nine C. betulus treatments and two of which was then mixed into the potting substrates. the six C. avellana treatments were examined at The original substrate was not removed because the 4.5 months because these species broke dormancy root systems were very dense. The seedlings were later, and very few short roots susceptible to mycor- repotted in 310 cm3 containers using either inoculated MTOS or peat:perlite. As controls, 15 Q. rhizal colonization had yet grown into the inoculated robur, six C. avellana and 10 C. betulus seedlings were substrate. Soil samples were taken from both sub- repotted into non-inoculated MTOS. All seedlings strates at 4.5 months to determine pH. All seedlings were watered manually with non-chlorinated tap were examined for truffle mycorrhizae 11 months water (pH 6.1) when dry and kept in a greenhouse after inoculation. (208C, 16 h light). They were treated against aphids Seedlings with T. aestivum mycorrhizae from the (Pyrsol, Bayer Environmental Science) once. The inoculation pilot study were planted on Gotland in break of dormancy was recorded by observing green November 2001 and June 2002. Roots were sampled Truffle cultivation in Sweden 47 in 2002Á2004 as described for the earlier plantations to investigate mycorrhizal development in the field. The results from the inoculation pilot study were statistically analysed by binomial tests, where all treatments were compared pairwise. The two possible outcomes in each treatment were success (presence of T. aestivum mycorrhiza) and failure (no T. aestivum mycorrhiza). The parameter assessed is the probability of success (p) in an individual trial, and the null hypothesis is that it is the same in the two treatments compared. Since there were enough trials in each treatment, a normal distribution approxima- tion of the binomial distribution was used. No correction for multiple tests was made and the significance may therefore be inflated. Figure 4. The first cultivated truffle in Scandinavia: a Burgundy truffle, Tuber aestivum, with mature spores visible as the brown tissue of the section. (Photograph: Eric Danell.) Results Fruit body production This small species is native to Scandinavia. Tub er rufum was subsequently found in truffle orchard 2A In May 2004, eight of the original 10 truffle orchards in October 2006. established in 1999 remained (Table III). Two On 29 November 2005, the first T. aestivum truffle orchards were lost when the majority of the seedlings produced in a Swedish truffle orchard was found in died owing to competition from ground vegetation. truffle orchard 2A in Fro¨jel parish (Figure 3, Table Of the eight remaining orchards, truffle orchard 2A III and Va). The fruit body grew a few centimetres had developed truffle burns (i.e. bruˆle´s). Burns are below ground, 20 cm from the stem of an oak. It areas with reduced vascular plant growth surround- contained mature spores and weighed 15 g (Figure ing truffle-infected trees due to the effect of the T. aestivum mycelium (Chevalier & Frochot, 1997a). In 4). The ground had been covered with chipped October 2004, T. rufum (the red truffle) was found in conifer bark for weed control. Exactly 1 year later, a four (3A, 5A, 7A and 9A) of these eight experi- second T. aestivum fruit body was found in truffle mental truffle orchards, indicating that the seedlings orchard 2A. Slightly smaller and still immature, it were large enough to support fruit body production. was also found about 20 cm from the stem of an oak,

Figure 3. Truffle orchard at Mulde Fritidsby, Fro¨jel parish, Gotland, Sweden, in 2006. The orchard was established in 1999 when 12 Quercus robur and 12 Corylus avellana seedlings inoculated with the Burgundy truffle, Tuber aestivum, were planted. (Photograph: Christina Wedeń.) 48 C. Wedeń et al. about 10 cm below the soil surface. The ground healthy, seedlings with a thin layer of chipped bark as around this tree had been covered with hay mulch. a weed suppressor were smaller (0.5Á1.5 m high) but In 2007, two T. aestivum fruit bodies were found still healthy looking, and seedlings where no weed under yet another oak. The ground cover was plastic suppressor was applied and no manual weeding done weave. The following week two more fruit bodies were very small and stunted (B 0.5 m high) or had † were found under this tree, and three additional fruit died. In this truffle orchard the herbicide Roundup bodies were found under the oak that produced the (Monsanto CropSciences, Sweden) was used once first truffle in 2005. All fruit bodies each weighed during the first year (seedlings covered in thick paper 10Á15 g each. bags during treatment). No effect was detected of the Roundup treatment on the occurrence of Experimental truffle orchards: mycorrhiza development, T. aestivum mycorrhizae, which was continuously seedling survival and truffle orchard management good at this site. Net cages were set up around seedlings to prevent Tuber aestivum mycorrhizae were found in all 10 grazing by hares and rabbits. Where rabbits or hares experimental truffle orchards 15 months after estab- bit off the stems of the seedlings only once, seedlings lishment in 1999. The seedlings were irrigated were stunted but survived. during June, July and August 1999 to ensure survival, but grew only marginally during their first growing season. Most of the seedlings in two of the Commercial truffle orchards: mycorrhiza development, truffle orchards died owing to competition from seedling survival and truffle orchard management weeds and grass (Table III, truffle orchards 1A and Fourteen truffle orchards planted in 2000 and 2001 10A). In the eight remaining truffle orchards, with T. aestivum-inoculated Q. robur seedlings were T. aestivum mycorrhizae were found yearly in followed in this study. Tuber aestivum mycorrhizae 2000Á2006. These truffle orchards remained in were found in all 14 truffle orchards investigated in good condition 7 years after establishment. There 2001Á2004. In one truffle orchard (15B) (Table V), were few or no weeds 1Á2 m around each seedling T. aestivum mycorrhizae could not be found with and the tree survival ranged from 79 to 96% (mean the soil core probing method in 2004 and therefore 89%). Tree growth varied among sites (Table III). further investigations were carried out. The presence The powdery mildew, Microsphaera alphitoides, has of T. aestivum mycorrhizae was confirmed instead by been observed on Q. robur seedlings in all truffle collecting mycorrhizae 0.25 m below ground, by orchards. One grower used the sulphur compound digging a short ditch along the side of a seedling. Kumulus DF (BASF AB) to reduce the infection Seedling survival in the commercial truffle orchard (truffle orchard 3A). Despite this treatment truffle was 85% after 4 years. Two main management mycorrhizae continued to be detected in truffle regimes were practised: (1) keeping the whole truffle orchard 3A. Mulching with chipped bark suppressed weeds orchard weed free and harrowing between the well when applied in a layer of 20 cm or more, but seedling rows; or (2) no management of weeds sometimes also supported new weed species which between the rows, or occasional mowing of the grass had not grown previously at a particular site. Where and weeds. In addition, cardboard discs (40 cm straw was used, it had to be reapplied every 2 years diameter) were applied around the base of the to suppress weed growth efficiently. At one very seedlings to suppress weed growth. Weed control windy site, the plastic weave battered the seedlings proved to be essential for seedling growth (Table III and had to be removed within the first year (Table and IV). The presence of T. aestivum mycorrhizae III, truffle orchard 5A). Several landowners reported did not differ between different managing regimes, that the white plastic weave created a greenhouse but mycorrhizae were much harder to find among effect, permitting weeds to grow beneath it. One the dense weed roots in non-weeded truffle orchards. landowner removed the plastic within the first year The cardboard discs used for weed control were too because weeds lifted the plastic, again endangering small to suppress weeds effectively. In some soils, the the seedlings (Table III, truffle orchard 6A). Others cardboard also favoured ant communities of differ- reported that the weeds growing under the plastic ent species, undermining the soil around the seed- were weaker and did not grow up again after ling root system. Powdery mildew (M. alphitoides) stepping on the plastic. In these four sites, plastic was observed on oaks in all truffle orchards. Tall suppressed weeds better than chipped bark and weeds and grass could sometimes act as a barrier to straw. This was well illustrated in one experimental infection, but competition from weeds seemed to truffle orchard (8A), where the seedlings growing have a more negative effect on seedling growth than with plastic weave were above 1.5 m high and very M. alphitoides infection. Truffle cultivation in Sweden 49

Soil composition and Tuber aestivum mycorrhiza perlite and 7.0Á7.5 in the MTOS, 4.5 months after abundance inoculation. Eleven months after inoculation, all seedlings were This evaluation includes both the eight surviving examined. Tuber aestivum mycorrhizae were found experimental truffle orchards and the 14 commercial on 34% of the Q. robur, C. avellana and C. betulus truffle orchards. All four truffle orchards with a seedlings, the success rate ranging from 0 to 78% negative mycorrhizal development trend as perceived depending on the seedling species, inoculation by the probing method (11B, 15B, 17B and 24B) treatment and substrate (Table VI). No T. aestivum deviated from soils in natural T. aestivum sites mycorrhizae were found on the 31 uninoculated (Wedeń et al., 2004a) by having a higher sand control seedlings. MTOS was significantly better content (Table Vb). Two of these truffle orchards than peat:perlite for Q. robur seedlings inoculated (15B and 17B) had pH values 0.3Á0.4 below the 5 with 510 spores per seedling (pB0.05). Quercus lowest value of the natural range, and two truffle robur was significantly better than C. betulus in orchards (11B and 15B) had exchangeable calcium 6 MTOS inoculated with 510 spores per seedling values less than half of the lowest value of the natural (pB0.05). Higher spore concentrations were signifi- range (Table Vb). In the orchard where T. aestivum cantly better than lower spore concentrations in mycorrhizae were not found by the probing method three cases (C. betulus in peat:perlite, 5107 spores in 2004 (15B), all three of these parameters were per seedling better than 5105; C. betulus in combined. Yet, even in this orchard, T. aestivum MTOS, 5107 spores per seedling better than 5 mycorrhizae were found with more extensive prob- 106; Q. robur in peat:perlite, 5106 spores per ing. Three out of 18 truffle orchards with easily seedling better than 5105)(pB0.05). No signifi- found mycorrhizae (9A, 13B and 14B) also deviated cant differences were observed from inoculating with strongly with respect to soil particle size distribution, mechanically broken asci or by dipping the roots in a but 15 out of 18 truffle orchards with easily found spore suspension before potting in an inoculated mycorrhiza had a soil particle size distribution within substrate. The statistics should be interpreted with the natural range. In the eight commercial truffle care, owing to the late break of dormancy of the orchards where T. aestivum mycorrhizae were found C. avellana and C. betulus seedlings relative the Q. in clusters with pronounced mycelial tufts (cystidae) robur seedlings in this experiment. radiating from the tip of the mycorrhiza, soil para- Morphological identification of T. aestivum mycor- meters were congruent with natural T. aestivum soils rhiza was confirmed by G. Chevalier (INRA, Cler- (Table Va). mont-Ferrand) and A. Zambonelli (Universita`di Bologna). Inoculation of greenhouse seedlings Budbreak began 1 month after inoculation in the Survival of inoculated Tuber aestivum seedlings in the Q. robur seedlings, 2 months after inoculation in the field C. betulus seedlings and 3 months after inoculation in In total 22 Q. robur, C. avellana and C. betulus the C. avellana seedlings. When the seedlings were seedlings which had established T. aestivum mycor- checked for mycorrhiza 4.5 months after inocula- rhizae in the inoculation experiment, when checked tion, the roots of the Q. robur seedlings had grown in October 2001, were planted on Gotland in into the T. aestivum-infested substrate, whereas November 2001 and in June 2002. Seedlings of all C. avellana and C. betulus seedlings still had very three species were planted in the experimental truffle few roots extended into the infested substrate. orchards to replace dead seedlings. One separate Tuber aestivum mycorrhizae were found on orchard was also established with 15 of the Q. robur four Q. robur seedlings from two different treatments seedlings. Mycorrhizae were sampled in 2004 and 4.5 months after inoculation (Table VI). All four T. aestivum mycorrhizae were still present in root Q. robur seedlings with T. aestivum mycorrhizae had samples from seedlings of all three tree species. been inoculated with mechanically broken asci and had been grown in MTOS (Table VI). Tuber aestivum Discussion mycorrhizae were not found on the Q. robur control seedlings. Only two C. avellana and five C. betulus In 2005, 6 years after planting, the first T. aestivum treatments were checked after 4.5 months, because fruit body was found in a Swedish truffle orchard. only sparse root development and no mycorrhizae This is the first cultivated truffle in Scandinavia. In were found. Therefore, the control seedlings were France, fruit bodies of T. aestivum are expected 5Á10 not examined at all at this stage. The substrate pH years after plantation (Chevalier & Frochot, 1997a). (pH-Fix, Macherey-Nagel) was 7.5Á8.0 in the peat:- Because of the northerly climate, and hence a 50 C. Wedeń et al. shorter growing season in Sweden, this time had Frochot, 1997a; Wedeń et al., 2004a). A mix of been anticipated to be longer than in France, but the Q. robur, C. avellana and C. betulus would be suitable results show fruit body production within the time in truffle orchards in Sweden, as C. avellana and expected in French truffle orchards. C. betulus will not compete with Q. robur for light and In this study, mycorrhizal seedlings were produced canopy space, but still ensure a shaded environment. using Swedish Q. robur, C. betulus and C. avellana Regardless of soil pH and other soil characteristics and Swedish T. aestivum inoculum. To the authors’ (Table Va, b), prolific mycorrhizae were found yearly knowledge, this is the first report of mycorrhizal in all truffle orchards on Gotland. A declining synthesis between T. aestivum and C. betulus, and the T. aestivum mycorrhiza development, as perceived first report of T. aestivum fruiting in association with from root probing, was linked to high sand content inoculated Q. robur seedlings. By analysis of root (87%), low amounts of exchangeable calcium samples in the four truffle orchards where these (B0.16%) and/or low pH (6.5 and below) (Table seedlings were planted in 2001Á2002, survival of Va, b). It could be that in siltier soils mycorrhizae are T. aestivum mycorrhizae was confirmed on all three easier to find because they are actually more seedling species 3 years after planting. In 2004, abundant. It could also be that the soil core probing T. aestivum mycorrhizae were present in all of the disfavours the sandier soils, since vital T. aestivum 22 studied truffle orchards on Gotland established in mycorrhiza clusters were found after a more thor- 1999Á2001. Additional root sampling in 2005 and ough investigation at the site where probing failed in 2006 in the eight oldest truffle orchards (planted in 2004, indicating that in the sandy soils short roots 1999) confirmed the continued presence of and mycorrhizae were sparse down to the depth of T. aestivum mycorrhizae in all these truffle orchards. up to 20 cm. Weed control around the seedlings does This paper also reports harvesting in late November not seem to affect the presence of the mycorrhizae 2005Á2007 of the first T. aestivum truffles produced during the first 5Á7 years after planting, but had a by inoculated Q. robur seedlings. This study will very strong effect on seedling growth (Table III and form the basis for future scientific and socioeco- IV). Mycorrhizal samples were harder to collect from nomic analyses of truffle biology and cultivation on trees surrounded by weeds and grass. This was due Gotland. in part to the disturbance of the weed roots, e.g. The risk of contamination by other ectomycor- Elytrigia repens, during root sampling, partly since rhizal fungi has been considered a problem in truffle the short roots seemed to develop deeper down in seedling production (Mamoun & Olivier, 1996; Hall the soil because of the competition for water by the & Wang, 1998). Mamoun and Olivier (1997) dense weed roots. showed that soil sterilization had a positive effect In 2005, the first T. aestivum fruit body was found on qualitative aspects of mycorrhizal colonization. under chipped bark mulch, in 2006 under hay mulch The present authors also recommend using sterilized and in 2007 under plastic weave. Weed control in the soil when inoculating in the future, to ensure the form of soil coverage, mulching, may help to retain production of seedlings with root systems well soil moisture and have a positive effect on T. aestivum colonized by T. aestivum. It is also possible that by proliferation in Swedish truffle orchards. Zambonelli using sterilized soil for inoculation, less truffle et al. (2005) reported straw mulching to have a inoculum is needed. In this experiment, the presence negative effect on T. aestivum mycorrhiza, because of of T. aestivum mycorrhizae increased with spore moisture retention by the straw, thereby increasing concentration, while in other studies where sterilized root growth and favouring competing mycorrhizal soil has been used, spore concentration has been development. In Sweden, however, straw and other shown not to be significant (Zambonelli & Bran- mulches may instead help to retain prolific zanti, 1989; Mamoun & Olivier, 1996; Fischer & T. aestivum development, since the cold and dry Colinas, 1996). Mechanical breaking of the spore Swedish climate (Wedeń et al., 2004a) could elim- sacs was not important, which could be due to the inate the causes of the negative effects observed in rapid breakdown of the spore ascus by microorgan- Italy. During the present truffle inventories, isms in the unsterilized soil/substrate. Natural soil is T. aestivum fruit bodies were not found in sites a complex substrate difficult to reproduce. There- with tall grass (Wedeń et al., 2004a). This seems to fore, efforts to find suitable well-defined artificial suggest that unmanaged T. aestivum truffle orchards substrates should be made to ensure production of in sites with a high weed growth pressure may not uniformly well-colonized seedlings. come into production. It is important to apply High seedling density (500 seedlings haÁ1)in knowledge about T. aestivum ecology to the truffle truffle orchards tends to result in earlier truffle orchards since just planting mycorrhizal seedlings production (Hall et al., 2007) and T. aestivum is is no guarantee of fruit body production. Results also often found in shaded biotopes (Chevalier & from the truffle orchards may also help in the Truffle cultivation in Sweden 51 understanding of the ecological requirements of and year would therefore give a yearly income of T. aestivum. This investigation has shown that approximately t3500, and a harvest of 40 kg haÁ1 T. aestivum fruit body formation is not dependent would give an income of t14,000 per year. on the long host tree continuity observed in natural From an ecotype perspective, it will be interesting sites on Gotland, strengthening the conclusion that to compare the development of the Swedish truffle T. aestivum biotopes are declining in Sweden orchards of French truffle strains planted in 1999, (Ga¨rdenfors, 2005). However, the old trees and with the truffle orchards of Swedish truffle strains long host tree continuity in the majority of planted in 2000Á2001, and 2006 and onwards. The T. aestivum sites on Gotland are promising for future finding of only one immature fruit body late in the long-term production in established truffle orchards. season 2006 could be an indication that French Tuber rufum (red truffle) fruit bodies have been truffle strains are not adapted to the Swedish found in five of the eight oldest truffle orchards climate. Seedlings inoculated with T. aestivum from including the truffle orchard producing T. aestivum Gotland have also been planted in the USA (Mis- fruit bodies in 2005 and 2006. Tuber rufum is a well- souri) (Pruett et al., 2004), New Zealand (North known species in Sweden (Bohus-Jensen, 1989) and Canterbury and Otago, South Island) and France also occurs in wild T. aestivum stands in Sweden, but (Champagne). The finding of the first cultivated seems to start maturing earlier in the season and to truffle 6 years after planting the truffle orchard is have a broader biotope range (unpublished data). comparable with French and New Zealand results in Also, in Italy, high Tuber magnatum (the white Alba Otago, and bodes well for the future establishment of truffle) fruit body yields occur in sites with very high new oak and hazel biotopes for truffle cultivation in proportions of T. rufum and T. maculatum mycor- Scandinavia. rhizal coexistence (Bertini et al., 2006). Soils with characteristics differing from natural sites may dis- favour fruit body production of T. aestivum or favour Acknowledgements competition from other ectomycorrhizal fungi, but at We are grateful to Ge´rard Chevalier and Alessandra this stage in the present study no such trends have be Zambonelli for mycorrhiza confirmation, and to seen. Ge´rard Chevalier for valuable advice on truffle As a consequence of the truffle research project, cultivation. We would like to thank Gunhild Beck- more than 5500 inoculated trees in more than 60 man, Ingvar Jakobsson, Bertil Widbom, Jonas Jo- truffle orchards have been planted on the islands of hansson, Nicklas Samils, Per Tjernby, Anders Gotland and O¨ land, in the counties of Va¨stergo¨tland, Wedeń and truffle orchard owners on Gotland for O¨ stergo¨tland and Ska˚ne on the Swedish mainland, contributing to this study. Mikael Thollesson kindly as well as in Finland, Denmark and Norway. Land- performed statistical analyses and Per Nystro¨m and owners were informed about the presence of natu- Inger Runeson, VegTech, were very helpful in the rally occurring truffles when found on their land. inoculation experiment. This study was financed by Two truffle cultivation societies (Gotlands tryffelfo¨r- the Municipality of Gotland, the European Agricul- ening and Rute Tryffel) and three truffle companies ture Guidance and Guarantee Fund under the ¨ ¨ (Gotlandstryffel AB, Olandstryffel and Ostergarn Objective 5b Gotland Programme, the European Tryffel) have been formed. Truffle dog training, Regional Development Fund under the Objective 2 truffle seedling production and processing are being Islands Programme and the Gotland County Ad- tested commercially. Wild T. aestivum was exported ministration, and further supported by Konsul Faxes from Gotland to France in 2003, when the long Fund, the KK Foundation, Sparbanksstiftelsen Alfa, summer drought in central and southern Europe led Carl the XVI Gustafs 50th Anniversary Fund and to depressed T. aestivum harvests there. During Carl Tryggers Foundation. 2004Á2007, wild Burgundy truffles from Gotland were sold at SEK 6600Á12,000 kgÁ1 in Stockholm. 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