Introduction

The fates of introduced mycorrhizal fungi in their new habitats has received considerable research attention in recent decades (see review by Vellinga et al. 2009). Reports in the literature have shown that fungal responses to new environments and new hosts as well as interactions with the local population varied. Introduced fungi can expand, infecting host-roots native to the introduction site, e.g., Laccaria fraterna (an Australian mycorrhizal fungus) and Cistus ladanifer L. in Spain (Diez 2005), and Amanita phalloides (a European mycorrhizal species) in North America (Wolfe et al. 2010). Alternatively, they may remain restricted to their original hosts for years, such as in the well-studied case of the American strain of Laccaria bicolor, S238 N, introduced into France along with Douglas fir (Selosse et al. 1998). Yet another potential outcome is replacement of the introduced mycorrhizal fungi by local fungal species that invade the host plant roots (e.g., Baciarelli-Falini et al. 2006; Chevalier et al. 1982; Garbaye et al. 1988).

The development of reliable inoculation procedures has made it possible to obtain seedlings of oak species and hazel (Corylus avellana L.) colonized by the black truffle of Périgord, T. melanosporum Vitt. (Chevalier et al. 1973; Palenzona 1969), paving the way for the establishment of oak groves for truffle production (Chevalier and Dupre 1990). Cultivation of T. melanosporum-colonized trees has since been attempted in areas and countries outside the natural habitat of the fungus. These efforts culminated in the harvesting of truffles in New Zealand (Hall 1996), the US (Garland 1999), and Australia (Elen 2007). A similar project aimed at introducing T. melanosporum into Israel was initiated in 1993 (Pinkas et al. 2000), and the first T. melanosporum mycorrhized seedlings were out-planted in 1994–1995 at several sites throughout Israel (Kagan-Zur et al. 2002).

The fate of T. melanosporum mycorrhiza associated with oak seedlings and hazelnut trees was followed about 4 years after out-planting (1998) at two very different sites in Israel. Most trees survived and T. melanosporum mycorrhizae (based on molecular analyses) survived on over 70% of the root sampled at both sites (Kagan-Zur et al. 2001). 1 year later, however, only a single T. melanosporum fruit body was collected between the two sites, (Kagan Zur et al. 2000). Subsequent examinations revealed no additional T. melanosporum fruit bodies. T. melanosporum mycorrhiza deteriorated continually until 2002 when no T. melanosporum mycorrhizas could be detected on sampled roots (Danai unpublished results). New T. melanosporum inoculated oak seedlings, some of which were, in fact, inoculated by T. aestivum Vitt., were planted in 1999 to refresh the grove.

In 2009 two fruit-bodies were found at one of the plantations during early spring. These were unripe, and were taken, at first, to be the long expected T. melanosporum. However, at this time of year T. melanosporum should be ripe or even over ripe. In 2010 a thorough study, aimed at unequivocally identify truffles collected at the same site, and assess yields was undertaken. We report here that the fruit bodies, collected under trees originally inoculated by T. melanosporum, were in fact T. aestivum. We also report that the yield encountered is comparable to yields of new plantations elsewhere.

This marks the first reported instance of an introduced mycorrhizal fungus being replaced by another introduced fungal species.

Materials and methods

Mycorrhizal tree plantation

One- to two-year-old colonized potted plants of native oak species, introduced European oak species, and hazelnuts were planted on Kibbutz Bar’am (Upper Galilee) during 1994–1995 and in 1999. The plantation setting was necessary to facilitate our identification of the most suitable host species for producing the best yields under our conditions. The different tree species were randomly planted within the grove. Trees were planted in rows at intervals of 4 m between trees and 6 m between rows. Soil analysis results were published elsewhere (Kagan-Zur et al. 2001).

The plot was irrigated during 1994–1998 using drip irrigation (3.2 lh−1, 1 m between drippers) every second day in summer and at greater time intervals in autumn and spring (correlated with soil moisture), and no water was supplied in winter. In total, about 3,500 m3 of water were supplied per year per hectare. From 1999 up to 2006, the drip system was replaced by sprinklers (one sprinkler between any two trees inside the rows) with the same irrigation regime. Irrigation was discontinued as of 2007, after which the trees depended solely on rainfall (average of 800 mm/year). The plot was superficially tilled yearly during springtime.

The first two fruit bodies were collected on 2009. In 2010, truffles were manually searched for by digging systematically around 125 trees in the plot.

Microscopic observations

Ripe fruit body pieces were ground in distilled water using a mortar and pestle, and samples were observed under a light microscope (Zeiss Standard 20, Germany).

DNA extraction

Approximately 100 mg of fruit body tissue samples were ground to a fine powder under liquid nitrogen using a mortar and pestle. The powder was then placed in a sterile 1.5 ml microcentrifuge tube. DNA was extracted with a kit following the manufacturer’s instructions (DNeasy Plant Mini Kit, Qiagen).

Internal Transribed Spacer (ITS) analyses as well as Hinf I and Hind III restriction enzyme reactions were performed as described elsewhere (Kagan-Zur et al. 1999).

Sequence analysis

DNA was extracted by the phenol–chloroform extraction procedure according to Henrion et al. (1994) from 50 mg freeze-dried tissue (gleba) crushed in liquid nitrogen. The primer pair used for Polymerase Chain Reaction was ITS1F/ITS4 (Sejalon-Delmas et al. 2000). The ITS DNA fragments were sequenced using the ITS1 primer (White et al. 1990) (one direction only). The sequence was deposited at the GenBank, accession no. JF770434.

Alignments and phylogenetic analysis

Several ITS sequences were retrieved from the National Center for Biotechnology Information for phylogenetic analysis (Table 1), and aligned using Clustal W 2.1 (http://www.clustal.org/download). An evolutionary tree based on these data was constructed along with 1,000 bootstrapping repeats by MEGA version 4.0.2 http://www.megasoftware.net/ (Kumar et al. 2008) using the neighbor-joining (NJ) method.

Table 1 Genbank accession numbers of truffles included in the phylogenetic tree presented in Fig. 3
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Results

In 2010 fruit bodies were collected (Fig. 1a) and studied. Their inner colors when cut (Fig. 1b) and spore morphologies observed under a light microscope (Fig. 1c) indicated that these fruit bodies had a greater resemblance to T. aestivum than to T. melanosporum.

Fig. 1
figure 1

Fruit bodies collected in the Bar’am grove. a An intact truffle, b a transverse cut of a fruit body, c the morphology of spores extracted from a fruit body. The spores were observed under a light microscope (×200)

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In an effort to definitively characterize the fruit bodies, molecular studies were then undertaken. An ITS-RFLP (Restriction Fragment Length Polymorphism) profile of Hinf I restriction fragments obtained from the fruit bodies was close, but not exactly identical, to that of certified T. melanosporum. However, Hind III restriction of the ITS fragments revealed a distinct difference between the T. melanosporum profile and those of the local fruit bodies, which resembled T. aestivum profiles (Fig. 2). Moreover, phylogenetic analysis of the sequenced ITS fragment positioned the Israeli tuber within the T. aestivum clade (Fig. 3).

Fig. 2
figure 2

ITS-RFLP analysis of amplified ITS fragments of T. melanosporum and T. aestivum compared to locally collected truffles. Lane 1, marker; HinfI cut: lane 2, T. melanosporum, lane 3, T. aestivum from France, lane 4, T. aestivum from Austria, lane 5, local T. aestivum. Hind III cut: lane 6, T. melanosporum, lane 7, T. aestivum from France, lane 8, T. aestivum from Austria, lane 9, local T. aestivum

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Fig. 3
figure 3

Phylogenetic tree of ITS sequences obtained from GenBank (Table 1), placing the Israeli truffle within the Tuber aestivum clade. The tree was constructed using the Neighbor-Joining method. Bar represents 0.05 changes

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During the spring of 2010, three organized searches recovered a total of 3.3 kg of fruit bodies for a calculated yield of about 6 kg/acre. The fruit bodies were collected in February, March, and April (Fig. 4). The average weight of truffles collected in February was only 15 g (Fig. 4), and the inner color of the gleba was pale white without the typical netting of dark veins. Examinations of their spore morphologies under a light microscope revealed that they were unripe—white in color and lacking ornamentation (data not shown). The fruit bodies reached maturity in April (average weight about 35 g), and the largest fruit body weighed 218 g (data not shown).

Fig. 4
figure 4

Average weight of Tuber aestivum fruit bodies, showing growth rate and development. Fruit bodies were collected from February until April when the fruits reached full size and ripeness. Standard error was calculated but not added to the figure as the size range was high, and the differences are not significant

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Not all the trees in the grove yielded fruit bodies, and some tree species produced more than others. For example, no fruit bodies were collected near any Quercus hartwissiana tree, while 50% of Coryllus avellana and Quercus calliprinos trees had at least one truffle at their roots (Fig. 5).

Fig. 5
figure 5

Yield distribution between the different oak species. The plot comprised local oak species (underlined), introduced oak species, and hazelnut trees

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Discussion

Attempts to establish T. melanosporum yielding plantations using nursery-inoculated seedlings started about four decades ago. However, the introduction of the fungus into habitats containing inocula of other truffles sometimes resulted in the replacement of the T. melanosporum mycorrhizae by that of the other native truffles (Baciarelli-Falini et al. 2006; Chevalier et al. 1982). Disinfecting the soil prior to the out-planting of T. melanosporum inoculated seedlings met with limited success (Frochot et al. 1990; Sourzat et al. 1990).

Drastic measures had to be undertaken prior to out-planting at sites where unfavorable habitat constraints limited T. melanosporum growth. These measures included elevation of soil pH and addition of calcium (Hall and Wang 2003), sometimes as pure lime (Garland 2011) to meet T. melanosporum requirements. Such measures simultaneously reduce competition from local mycorrhizal fungi (Hall and Wang 2003). In the case reported here, although the area where the grove was planted was carefully chosen to meet the known requirements of climate and soil conditions for cultivating T. melanosporum, a reduction in the inoculation levels on oak roots was observed 4 years after outplanting (Kagan-Zur et al. 2001), an outcome probably due to some undefined environmental limiting condition(s) to the host, to the fungus, or to both. T. aestivum, on the other hand, is a more robust species (Pruett 2008). It was recently introduced into foreign habitats such as Finland (Shamekh et al. 2009) and the USA (Pruett et al. 2008). In both habitats, researchers report T. aestivum mycorrhizae to be thriving.

Our physical and microscopic analyses indicated that the truffles unearthed at the Bar’am grove might be T. aestivum (Fig. 1). The RFLP molecular study (Fig. 2) corroborated this asumption, while the phylogenetic placing of the Israeli truffles in the T. aestivum clade (Fig. 3), clearly identified the truffles collected as T. aestivum, not the expected T. melanosporum. It seems that T. melanosporum mycorrhizae deteriorated slowly from the day of seedling out-planting on 1994/1995, surviving on roots of only 70% of the trees 4 years later (Kagan-Zur et al. 2001) and declining yearly ever since, completely disappearing on 2002 (unpublished), eventually leaving the trees devoid of suitable mycorrhizal associations. Inadvertently introduced into the grove during 1999, T. aestivum filled this need and with time replaced the original T. melanosporum mycorrhizae, a process that culminated in the production of T. aestivum fruit bodies. It should be noted that fruit bodies were found under trees planted during 1994/5, while T. aestivum was only introduced in 1999, indicating that inoculation of existing non-inoculated, fully developed groves (5 years old) is possible under our conditions.

No reports of finding T. aestivum in Israel exist. All known local truffles belong to the Pezizaceae family (desert truffles) (Binyamini 1980; Kagan-Zur and Roth-Bejerano 2009). Although it cannot be categorically ruled out that the T. aestivum which replaced the T. melanosporum inoculated grove was local, no truffles at all were ever collected at the roots of oak species in nearby natural groves. This finding indicates that within the short time span of 10 years, accidentally introduced T. aestivum almost totally replaced T. melanosporum mycorrhizae, creating a T. aestivum truffle-yielding plantation.

Native oak species were unexpectedly found to be better hosts for T. aestivum than the introduced oak species, which were intentionally chosen for introduction since they are known natural hosts of T. melanosporum. However, these foreign oak species may have encountered difficulties in their new habitat, thus failing to support mycorrhiza of any of the introduced fungi.

Under our climate conditions, the fruiting season of T. aestivum precedes that in Europe by several weeks, as reflected in its rate of fruit body development, and that should create an opportunity to extend the marketing season (Fig. 4). T. aestivum yields were comparable to those reported for young groves elsewhere (Southern Woods Nursery 2011).

In conclusion, the environmental conditions on this Upper Galilee plantation, though unsuitable for T. melanosporum, fit the requirements of the more robust T. aestivum fungus, which thrives on local oak species.